Metabolic engineering for microbial production of terpenoid products

ABSTRACT

The invention relates to methods and bacterial strains for making terpene and terpenoid products, the bacterial strains having improved carbon pull through the MEP pathway and to a downstream recombinant synthesis pathway.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/450,707 filed Jan. 26, 2017, the content of which is herebyincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 26, 2018, isnamed MAN-009PC_ST25 and is 125,103 bytes in size.

BACKGROUND

The food and beverage industries as well as other industries such as theperfume, cosmetic and health care industries routinely use terpenesand/or terpenoid products, including for use as flavors and fragrances.However, factors such as: (i) the availability and high price of theplant raw material; (ii) the relatively low terpene content in plant;and (iii) the tedious and inefficient extraction processes to producesufficient quantities of terpene products on an industrial scale allhave stimulated research on the biosynthesis of terpenes usingplant-independent systems. Consequently, effort has been expended indeveloping technologies to engineer microorganisms for convertingrenewable resources such as glucose into terpenoid products. Bycomparison with traditional methods, microorganisms have the advantageof fast growth without the need for land to sustain development.

There are two major biosynthetic routes for the essential isoprenoidprecursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate(DMAPP), the mevalonate (MVA) pathway and the methylerythritol phosphate(MEP) pathway. The MVA pathway is found in most eukaryotes, archaea anda few eubacteria. The MEP pathway is found in eubacteria, thechloroplasts of plants, cyanobacteria, algae and apicomplexan parasites.E. coli and other Gram-negative bacteria utilize the MEP pathway tosynthesize IPP and DMAPP metabolic precursors. While the MEP pathwayprovides a theoretically better stoichiometric yield over the MVApathway, the MEP pathway in E. coli and in other bacteria has a varietyof intrinsic regulation mechanisms that control and/or limit carbon fluxthrough the pathway. See, Zhao et al., Methylerythritol PhosphatePathway of Isoprenoid Biosynthesis, Annu Rev. Biochem. 2013; 82:497-530;Ajikumar P K, et al., Isoprenoid pathway optimization for Taxolprecursor overproduction in Escherichia coli. Science 2010; 330-70-74.

Microbial strains and methods for improving carbon flux through the MEPpathway and through recombinant downstream terpene and terpenoidsynthesis pathways are needed for industrial-scale production ofterpenes and terpenoids in bacterial systems.

SUMMARY OF THE INVENTION

In various aspects, the invention relates to methods and bacterialstrains for making terpene and terpenoid products. In certain aspects,the invention provides bacterial strains with improved carbon flux intothe MEP pathway and to a downstream recombinant synthesis pathway, tothereby increase terpene and/or terpenoid production by fermentationwith inexpensive carbon sources (e.g., glucose).

In some aspects, the invention relates to bacterial strains thatoverexpress IspG and IspH, so as to provide increased carbon flux to1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) intermediate, butwith balanced expression to prevent accumulation of HMBPP at an amountthat reduces cell growth or viability, or at an amount that inhibits MEPpathway flux and/or terpenoid production. Increasing expression of bothIspG and IspH significantly increases titers of terpene and terpenoidproducts. In contrast, overexpression of IspG alone results in growthdefects, while overexpression of IspH alone does not significantlyimpact product titer. HMBPP metabolite can act as a regulator orinhibitor of the MEP pathway, and may be toxic to the bacterial cells atcertain levels. For example, in some embodiments, HMBPP does notaccumulate at more than about 10 mg/g dry cell weight (DCW), or in someembodiments does not accumulate at more than about 5 mg/g of DCW, or atmore than about 2 mg/g DCW. Thus, the balanced overexpression of IspGand IspH (e.g., favoring more IspH activity) is important to pull MEPcarbon downstream through HMBPP to IPP while preventing its imbalanceand accumulation.

In various embodiments, the bacterial strain overexpresses a balancedMEP pathway to move MEP carbon to the MEcPP intermediate, the substratefor IspG, and includes one or more genetic modifications to support theactivities of IspG and IspH enzymes, which are Fe-sulfur clusterenzymes. Exemplary modifications include those that enhance the supplyand transfer of electrons through the MEP pathway, and/or to terpene orterpenoid products. These include recombinant expression of one or moreoxidoreductase enzymes, including oxidoreductases that oxidize pyruvateand/or lead to reduction of ferredoxin (which supplies electrons to theMEP pathway). An exemplary oxidoreductase is E. coli YdbK and orthologsand derivatives thereof.

In various embodiments, the microbial strain comprises an overexpressionof or complementation with one or more of a flavodoxin (fldA),flavodoxin reductase, ferredoxin (fdx), and ferredoxin reductase.

In other aspects, the invention provides bacterial strains thatoverexpress PgpB or NudB, which dephosphorylate FPP to farnesol, and IPPand DMAPP to isoprenol and prenol, respectively. In these embodiments,the cell contains an additional product pull on the MEP pathway, whiledraining excess MEP carbon from the pathway outside the cell, andthereby avoiding intrinsic feedback inhibition mechanisms. Further,since these products accumulate outside the cell, they can be used totrack carbon flux through the MEP pathway, even without a downstreamterpenoid synthesis pathway installed. Thus, bacteria strainsoverexpressing PgpB and/or NudB are convenient tools for balancing theexpression of MEP pathway genes. Additionally, or alternatively, in someembodiments, the bacterial strain overexpresses one or more strongsynthases with sufficient product pull on the MEP pathway to avoidintrinsic feedback inhibition mechanisms. By way of example, in someembodiments, the synthase is Artemisia annua farnesene synthase.

For production of terpene or terpenoid product, the bacterial cell willcontain a recombinant downstream pathway that produces the terpenoidfrom IPP and DMAPP precursors. In certain embodiments, the bacterialcell produces one or more terpenoid compounds, such as monoterpenoids,sesquiterpenoids, triterpenoids, and diterpenoids, among others. Suchterpenoid compounds find use in perfumery (e.g. patchoulol), in theflavor industry (e.g., nootkatone), as sweeteners (e.g., steviolglycosides), as colorants, or as therapeutic agents (e.g., taxol).

The recovered terpene or terpenoid may be incorporated into a product(e.g., a consumer or industrial product). For example, the product maybe a flavor product, a fragrance product, a sweetener, a cosmetic, acleaning product, a detergent or soap, or a pest control product. Thehigher yields produced in embodiments of the invention can providesignificant cost advantages as well as sustainability and qualitycontrol of the terpene or terpenoid ingredient.

Other aspects and embodiments of the invention will be apparent from thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic for terpenoid production through the MEP pathway.A bacterial cell is represented, taking in glucose as a carbon source.Glucose is converted to biomass through the TCA cycle or funneledthrough the MEP pathway to the desired terpenoid products. Glucose comesinto the cell and is converted to pyruvate (PYR) with glyceraldehyde3-phosphate as an intermediate (GAP). PYR and GAP are combined to makeDOXP, which is converted to MEP and commits the pathway to FPP (goingthrough MEcPP). DOX and ME are dephosphorylated products of DOXP andMEP, respectively. DOX, ME, and MEcPP are found outside the cell. Themore flux that is forced into the MEP pathway, the more these productsare found extracellularly. These side products can be used as markers ofbottlenecks in the MEP pathway, and to identify targets for engineering.By overexpressing nudB or pgpB, IPP and DMAPP or FPP aredephosphorylated to prenol and isoprenol or farnesol, respectively,which accumulate outside the cell and can be used to alleviateintermediate accumulation and the activation of feedback inhibition ofthe MEP pathway. Black arrows show enzyme-mediated biochemical reactionstowards terpenoids, light grey arrows show a competing side product,dark grey arrows show transport of a product outside of the cell, andwhite arrows show condensed pathways for simplicity.

FIG. 2 shows that increasing expression of ispH alone or ispH and ispGtogether improve terpenoid product titers in strains engineered toincrease the amount of carbon entering the MEP pathway, but ispG alonedecreases productivity. The control strain is an E. coli strain withadditional copies of dxs, dxr, ispD, ispE, ispF, and idi (no additionalcopies of ispG or ispH), among other changes to improve MEP pathwayflux. Strains include a 20 kb deletion, which was not engineered.

FIG. 3 shows that increasing ispG and/or ispH expression in modifiedproduction strains with enhanced MEP pathways impacts the MEP productdistribution pattern. Upper panel (1× scale) shows all MEP pathwaymetabolites, with the majority of products being DOX, ME, and MEcPP.Middle panel (100× scale), shows MEP pathway metabolites with DOX, ME,and MEcPP not reported; in this case, DOXP and MEP are the mostrepresented. Lower panel (25000× scale) shows only HMBPP concentration.In these panels, total extracellular and intracellular metabolites areshown from extracted cultures (broth plus cells), such that the reportedconcentration is relative to volume of extract.

FIG. 4 shows the proportion of each individual MEP metabolite foundinside or outside the cell (‘Intra’ vs ‘Extra’). These values do notreflect absolute abundance, e.g. there is far more DOX in total thanthere is HMBPP. While DOX is 100% extracellular, HMBPP is 100%intracellular. The strain profiled in FIG. 4 is the ‘Control+ispH/ispG’top performing strain. DOXP/DOX, MEP/ME, and MEcPP accumulate almostentirely, if not entirely, in the extracellular medium, while CDP-ME,CDP-MEP, HMBPP, IPP/DMAPP, and FPP are observed 100% intracellularly.The percentage of each metabolite found intracellularly is shown at thetop of the graph.

FIG. 5 shows that uncompensated ispG upregulation causes a significantdrop in cell growth, as determined by UV absorbance at 600 nm. Whilesome changes to final cell density is observed in strains compensatedwith ispH or ispH and ispG together, the variation is not significant.

FIG. 6 shows that overexpression of pgpB can triple farnesol titers instrains engineered to enhance flux through MEP pathway, but without adownstream terpenoid product pathway installed. The control strain hasadditional copies of dxs, dxr, ispD, ispF, ispE, ispG, ispH, and idiunder varying levels of constitutive expression, and also has ydbKoverexpressed. The strain accumulates moderate amounts of farnesol,presumably as ‘spill-over’ from too much FPP accumulation, which feedsback on the pathway, and suffers from markedly slower growth compared towild-type. When pgpB is overexpressed in this strain, the excess FPP ismore efficiently converted to farnesol (preventing feedback control) andthe flux is effectively pulled through the MEP pathway.

FIG. 7 shows that increasing and tuning expression of ispG′ and/or ispHin a strain that produces farnesol can improve product titer. Thecontrol strain has additional copies of dxs, dxr, ispD, ispF, ispE,ispG, ispH, and idi, as well as additional copies of ydbK and pgpB.Additional copies of ispH and/or ispG′ are integrated into the strainsunder increasing promoter strength (+, ++, +++).

FIG. 8 shows that increase in farnesol product titer (shown in FIG. 7)is accompanied by a decrease in MEcPP pool size, and depends on theratio of ispG and ispH.

FIG. 9 shows that idi overexpression increases product titer in a strainthat does not overexpress ispGH, and decreases titer in two strains thatdo overexpress ispGH, indicating that the balance between IPP and DMAPPcontrolled by Idi activity can be tuned up or down depending on theneeds of the downstream pathway.

FIG. 10 illustrates the role of YdbK as a pyruvate:flavodoxinoxidoreductase and/or pyruvate synthase in enhancing terpenoidbiosynthesis.

FIG. 11 shows that expressing an additional copy of ydbK underincreasing promoter strength can improve terpenoid production. Thecontrol strain produces terpenoid product A, and has additional copiesof genes dxs, dr, ispD, ispE, ispF, ispG′, ispH, and idi of the MEPpathway under defined constitutive expression.

FIG. 12 shows that improvements in terpenoid product titer from ydbKoverexpression requires sufficient ispG and/or ispH. Control A hasadditional copies of dxs, ispD, ispF, and idi of the MEP pathway, anon-engineered 20 kb deletion, as well as other modifications to improveperformance of iron-sulfur cluster proteins. Control B is Control A plusan additional integrated copy of ispG′ and ispH in operon configuration(G′ first, such that the H/G ratio favors G), while Control C is ControlA plus an additional integrated copy of ispH and ispG′ in operonconfiguration (H first, such that the H/G ratio favors H).

FIG. 13 shows that expressing fdx in addition to ydbK can improveterpenoid titers. The control strain produces terpenoid product A, andhas additional copies of genes ds, dxr, ispD, ispE, ispF, ispG′, ispH,and idi of the MEP pathway under defined constitutive expression. Thestrain also has a non-engineered 20 kb deletion and other modificationsto improve performance of iron-sulfur cluster proteins.

FIG. 14 illustrates the interface between glycolysis and the MEPpathway, and illustrates opportunities to tune co-factor availability byaltering expression of oxidoreductase enzymes.

FIG. 15 is a diagram illustrating the three known reactions in E. colito convert pyruvate (PYR) to acetyl-CoA (AcCoA) and illustrates howreducing or eliminating PDH mediated conversion of PYR to AcCoA resultsin the increase of PFOR-mediated conversion of PYR to AcCoA.

FIGS. 16A-D are graphs showing the fold change in terpenoid productproduction in bacterial strains having overexpressed YdbK and knockoutof aceE (ΔaceE), as compared to control. The control is the same strainwithout (ΔaceE). ΔaceE prevents PDH-mediated conversion of PYR to AcCoA.FIG. 16A shows the fold change in bacterial strains that produceterpenoid Product B. FIG. 16B shows the fold change in bacterial strainsthat produce terpenoid Product C. FIG. 16C shows the fold change inbacterial strains that produce terpenoid Product D. FIG. 16D shows thefold change in bacterial strains that produce terpenoid Product E.

FIG. 16E is a graph showing that bacterial strains that produceterpenoid Product D, overexpress YdbK, and have ΔaceE show a reductionin extracellular MEcPP as compared to control (no ΔaceE).

FIGS. 17A-C are graphs showing the fold change in terpenoid product inbacterial strains having overexpressed YdbK and mutated aceE (aceE mut),as compared to control (no aceE mut). aceE mut reduces PDH-mediatedconversion of PYR to AcCoA. FIG. 17A shows the fold change in bacterialstrains that produce terpenoid Product B. FIG. 17B shows the fold changein bacterial strains that produce terpenoid Product C. FIG. 17C showsthe fold change in bacterial strains that produce terpenoid Product D.

FIG. 17D is a graph showing that a bacterial strain that producesterpenoid Product D, overexpresses YdbK, and expresses an aceE mut has areduction in extracellular MEcPP as compared to control (no aceE mut).

FIG. 18 is a diagram illustrating the three known reactions in E. colito convert pyruvate (PYR) to acetyl-CoA (AcCoA) and illustrates howexpressing fdx or fldA homologs can increase the electron supply to IspGand/or IspH through the Fd redox reaction (shown in bold).

FIG. 19A is a graph showing the fold change (as compared to the emptyvector control (emp)) of terpenoid Product B production in a bacterialstrain engineered to overexpress YdbK and overexpress an fdx or fldAhomolog.

FIG. 19B is a graph showing the fold change (as compared to control) ofterpenoid Product D in a bacterial strain engineered to overexpress YdbKand Cv.fdx (an fdx homolog from Allochromatium vinosum).

FIG. 19C is a graph showing that a bacterial strain that producesterpenoid Product D and overexpresses YdbK and Cv.fdx has a reduction inextracellular MEcPP as compared to control (without overexpression ofCv.fdx).

FIG. 20 is a graph showing the fold change in the production ofterpenoid product in bacterial strains that produce terpenoid Product Fand which overexpress one or more PFOR or fpr homologs and, optionally,a fdx or fldA homolog.

DETAILED DESCRIPTION OF THE INVENTION

In various aspects, the invention relates to bacterial strains andmethods for making terpene and terpenoid products, the bacterial strainshaving improved carbon flux through the MEP pathway and to a downstreamrecombinant synthesis pathway. In various embodiments, the inventionprovides for increased terpene and/or terpenoid product yield byfermentation of the bacterial strains with carbon sources such asglucose, glycerol, sucrose, and others.

For example, in some aspects the invention provides a bacterial strainthat produces isopentenyl diphosphate (IPP) and dimethylallyldiphosphate (DMAPP) through the MEP pathway, and converts the IPP andDMAPP to a terpene or terpenoid product through a downstream synthesispathway. In the bacterial strain, IspG and IspH are overexpressed suchthat IspG activity and IspH activity are enhanced to provide increasedcarbon flux to 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP)intermediate, but balanced to prevent accumulation of HMBPP at an amountthat significantly reduces cell growth, viability, MEP pathway flux, orproduct titer.

Increasing expression of both IspG and IspH can significantly increasetiters of terpene and terpenoid products. Increasing expression of justIspG or IspH alone does not significantly improve titer. Further,overexpression of IspG alone can result in growth defects, which mayrelate to the observation that HMBPP (the intermediate in the MEPpathway produced by IspG, and consumed by IspH) is not foundextracellularly, but is found 100% intracellularly. HMBPP metaboliteappears to act as an inhibitor of the MEP pathway, and appears to betoxic to the bacterial cell at certain levels. Thus, the balance ofactivity between IspG and IspH is important to prevent HMBPP imbalanceand accumulation.

HMBPP accumulation can be determined as an amount per dry cell weight(DCW). For example, in some embodiments, HMBPP does not accumulate atmore than about 10 mg/g DCW, or in some embodiments does not accumulateat more than about 8 mg/g of DCW, or in some embodiments does notaccumulate at more than about 5 mg/g of DCW, or in some embodiments doesnot accumulate at more than about 4 mg/g DCW, or in some embodimentsdoes not accumulate at more than about 2 mg/g DCW. In some embodiments,HMBPP does not accumulate at more than about 1 mg/g DCW, or does notaccumulate at more than about 0.5 mg/g DCW, or more than about 0.2 mg/gDCW, or more than about 0.1 mg/g DCW. The balanced overexpression ofIspG and IspH (e.g., favoring more IspH activity) is important to pullMEP carbon downstream through HMBPP to IPP while preventing itsimbalance and accumulation.

In some embodiments, IspG and IspH are overexpressed by introducingrecombinant ispG and ispH genes into the bacterial strain. In otherembodiments, the endogenous genes can be overexpressed by modifying, forexample, the endogenous promoter or ribosomal binding site. Whenintroducing recombinant ispG and/or ispH genes, the genes may optionallycomprise one or more beneficial mutations.

In some embodiments, the additional gene may be substantially identicalto the wild-type enzyme (e.g., the E. coli wild-type enzyme), or may bemodified to increase activity or may be an IspG or IspH ortholog havingsimilar, higher, or lower activity than the native bacterial (e.g., E.coli) enzyme. For example, with respect to IspG, the amino acid sequencemay have 50% or more sequence identity with SEQ ID NO:1, or at leastabout 60% sequence identity, or at least about 70% sequence identity, orat least about 80% sequence identity, or at least about 90′ sequenceidentity, or at least about 95% sequence identity, or at least about 98%sequence identity with the amino acid sequence of SEQ ID NO:1. In someembodiments, from 1 to about 10, or from 1 to about 5 amino acidsubstitutions, deletions, and/or insertions are made to the IspG aminoacid sequence (SEQ ID NO:1) to alter the activity of the protein,including substitutions to one or more of the substrate binding site oractive site. Modifications to E. coli or other IspG can be informed byconstruction of a homology model. For example, a suitable homolog forconstruction of an E. coli IspG homology model is disclosed in: Lee M,et al. Biosynthesis of isoprenoids: crystal structure of the[4Fe-4S]cluster protein IspG. J Mol Biol. 2010 Dec. 10; 404(4):600-10.An exemplary IspG mutant with improvements in activity has four aminoacid substitutions with respect to the wild type E. coli enzyme(referred to herein as IspG′).

Further, with respect to IspH, the amino acid sequence may have 50% ormore sequence identity with SEQ ID NO:2, or at least about 60% sequenceidentity, or at least about 70% sequence identity, or at least about 80%sequence identity, or at least about 90% sequence identity, or at leastabout 95% sequence identity, or at least about 98% sequence identitywith the amino acid sequence of SEQ ID NO:2. In some embodiments, from 1to about 10, or from 1 to about 5, amino acid substitutions, deletions,and/or insertions are made to the IspH amino acid sequence (SEQ ID NO:2)to alter the activity of the protein, including substitutions to one ormore of the substrate binding site or active site. Modifications to theIspH enzyme can be informed by available IspH structures, includingGrawert, T., et al. Structure of active IspH enzyme from Escherichiacoli provides mechanistic insights into substrate reduction 2009 Angew.Chem. Int. Ed. Engl. 48: 5756-5759.

Table 1 provides a list of alternative enzymes useful for constructingbacterial strains and/or modifying IspG or IspH enzymes for enhancedexpression in bacterial cells or enhanced physical properties, each ofwhich can be modified by amino acid substitution, deletion, and/orinsertion. For example, the amino acid sequence may have 50% or moresequence identity, or at least about 60% sequence identity, or at leastabout 70% sequence identity, or at least about 80% sequence identity, orat least about 90′ sequence identity, or at least about 95% sequenceidentity, or at least about 98% sequence identity with an amino acidsequence described in Table 1. In some embodiments, from 1 to about 10,or from 1 to about 5, amino acid substitutions, deletions, and/orinsertions are made to a sequence of Table 1 to alter the activity ofthe protein, including substitutions to one or more of the substratebinding site or active site. In some embodiments, the IspG and/or IspHenzyme is an ortholog of the E. coli enzyme having improved propertiesor activity under conditions used for culturing.

TABLE 1 Gene Species Accession number ispG Bacillus subtilis NP_390386.1ispG Chloroboculum tepidum NP_661053.1 ispG Synechocystis sp. PCC 6803WP_010872347.1 ispH Bacillus subtilis NP_390395.2 ispH Burkholderia sp.MSh1 WP_031398482.1 ispH Chloroboculum tepidum NP_661187.1 ispH Steviarebaudiana ABB88836.2 ispH Stevia rebaudiana ALJ30091.1 ispHSynechocystis sp. PCC 6803 WP_010873388.1

The expression of the recombinant IspG and IspH enzymes can be balanced,for example, by modifying the promoter strength, gene copy number,position of the genes in an operon, and/or modifying the ribosomebinding site sequence of the ispG and/or ispH recombinant genes. Whenthe expression and/or activity of IspG and IspH are balanced, HMBPPintermediate does not accumulate in cells substantially more than in aparent strain that does not comprise the recombinant or modified ispGand ispH genes. This is despite the substantial increase in carbon fluxthrough the MEP pathway that is required for commercial production ofterpenes and terpenoids by fermentation. This result is shown in FIG. 3,where strains overexpressing ispG and ispH, which can produce close to a4-fold increase in product titer as compared to a control strain thatdoes not overexpress ispG and ispH (FIG. 2), nevertheless do notaccumulate HMBPP intermediate above that in the control.

In some embodiments, the activity and/or expression of recombinant IspHis higher than the activity and/or expression of the recombinant IspG.An IspG/IspH ratio that favors more H enzyme results in high fluxthrough the MEP pathway relative to a strain favoring the IspG side ofthe ratio. IspG and IspH work sequentially to convert MEcPP to HMBPP,then to IPP. Increasing IspG accumulates a larger HMBPP pool (which canshow inhibitory effects on strain growth), while increasing IspH shrinksthe HMBPP pool as it is converted to IPP. Thus, the ideal balancebetween IspG and IspH enhances the rate of both HMBPP formation andconsumption, while avoiding HMBPP accumulation, which significantlyimproves flux through the MEP pathway to the target terpenoid. A slightfavoring of IspH over IspG can further improve productivity by 25%, tonearly 4 times the titers of the parent strain. See FIG. 2.

Thus, in some embodiments, the expression of the recombinant IspH ishigher than the expression of the recombinant IspG. For example, therecombinant IspH and IspG enzymes can be expressed from an operon, withispH positioned before ispG in the operon. The gene positioned first inthe operon will be slightly favored for expression, providing an elegantbalancing mechanism for IspH and IspG. In some embodiments, ispG can bepositioned first, optionally together with other modifications, such asmutations to the RBS to reduce expression, or point mutations to one orboth of IspG and IspH that balance activity at the level of enzymeproductivity. In some embodiments, ispG and ispH are expressed inseparate operons (e.g., monocistronic) and expression balanced usingpromoters or RBSs of different strengths.

In some embodiments, IspH and IspG are expressed together from an operon(with the ispH gene positioned before the ispG gene), and with theoperon expressed under control of a strong promoter. While increasingpromoter strength has a positive impact on productivity when ispH ispositioned before ispG in the operon, increasing promoter strength canhave a negative impact when ispG is positioned before ispH. See FIG. 7,using farnesol production as a surrogate for product.

Recombinant IspG and IspH enzymes can be expressed from a plasmid or theencoding genes may be integrated into the chromosome, and can be presentin single or multiple copies, in some embodiments, for example, about 2copies, about 5 copies, or about 10 copies per cell. Copy number can becontrolled by use of plasmids with different copy number (as is wellknown in the art), or by incorporating multiple copies into the genome,e.g., by tandem gene duplication.

In some embodiments, the microbial strain has high flux through the MEPpathway, including for example, by overexpression of one or more MEPenzymes (e.g., in addition to IspG and IspH). With glucose as carbonsource, the theoretical maximum for carbon entering the MEP pathway isabout 30% in E. coli. Prior yields of MEP carbon reported in theliterature are less than 1%. See, Zhou K, Zou R, Stephanopoulos G, TooH-P (2012) Metabolite Profiling Identified MethylervthritolCyclodiphosphate Efflux as a Limiting Step in Microbial IsoprenoidProduction. PLoS ONE 7(11): e47513. doi:10.1371/journal.pone.0047513.Overexpression and balancing of MEP pathway genes, in addition to othermodifications described herein can pull carbon through the MEP pathwayand into a downstream synthesis pathway to improve carbon flux throughto terpene and/or terpenoid products.

The host cell (the bacterial strain) expresses an MEP pathway producingisopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP).Specifically, glucose comes into the cell and is converted to pyruvate(PYR) with glyceraldehyde-3-phosphate as an intermediate (G3P or GAP).G3P and PYR are combined to make 1-deoxy-D-xylulose-5-phosphate (DOXP),which is converted to 2-C-methyl-D-erythritol 4-phosphate (MEP) andcommits the pathway to IPP and DMAPP. DOX, ME, and MEcPP are foundoutside the cell. The more flux into the MEP pathway, the more theseproducts are found extracellularly in strains with unbalanced pathways.See FIG. 1.

The MEP (2-C-methyl-D-erythritol 4-phosphate) pathway is also called theMEP/DOXP (2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose5-phosphate) pathway or the non-mevalonate pathway or the mevalonicacid-independent pathway. The pathway typically involves action of thefollowing enzymes: 1-deoxy-D-xylulose-5-phosphate synthase (Dxs),1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr, or IspC),4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD),4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE),2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF),1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG),1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IspH) andisopentenyl diphosphate isomerase (Idi). The MEP pathway, and the genesand enzymes that make up the MEP pathway, are described in U.S. Pat. No.8,512,988, which is hereby incorporated by reference in its entirety.Thus, genes that make up the MEP pathway include dxs, dxr (or ispC),ispD, ispE, ispF, ispG, ispH, idi, and ispA. The amino acid sequencesfor MEP pathway enzymes are shown in the attached listing of Sequences.

IPP and DMAPP (the products of the MEP pathway) are the precursors ofterpenes and terpenoids, including monoterpenoids, sesquiterpenoids,triterpenoids, and diterpenoids, which have particular utility in theflavor, fragrance, cosmetics, and food sectors. Synthesis of terpenesand terpenoids proceeds via conversion of IPP and DMAPP precursors togeranyl diphosphate (GPP), farnesyl diphosphate (FPP), or geranylgeranyldiphosphate (GGPP), through the action of a prenyl transferase enzyme(e.g., GPPS, FPPS, or GGPPS). Such enzymes are known, and are describedfor example in U.S. Pat. No. 8,927,241, WO 2016/073740, and WO2016/029153, which are hereby incorporated by reference in theirentireties.

In various embodiments, the invention results in substantialimprovements in MEP carbon. As used herein, the term “MEP carbon” refersto the total carbon present as an input, intermediate, metabolite, orproduct of the MEP pathway. Metabolites include derivatives such asbreakdown products, and products of phosphorylation anddephosphorylation. MEP carbon includes products and intermediates ofdownstream pathways including terpenoid synthesis pathways. For purposesof this disclosure, MEP carbon includes the following inputs,intermediates, and metabolites of the MEP pathway: D-glyceraldehyde3-phosphate, pyruvate, 1-deoxy-D-xylulose-5-phosphate,1-deoxy-D-xylulose, 2-C-methyl-D-erythritol-5-phosphate,2-C-methyl-D-erythritol, 4-diphosphocytidyl-2-C-methyl-D-erythritol,2-phospho-4-diphosphocytidyl-2-C-methyl-D-erythritol,2C-methyl-D-erythritol 2,4-cyclodiphosphate,1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, isopentenyl diphosphate,and dimethylallyl diphosphate. MEP carbon further includes intermediatesand key metabolites in the downstream terpenoid synthesis pathwayexpressed by the cell. While the identity will vary based upon pathwayand enzymes employed, such products include: geranyl diphosphate (GPP),farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), orgeranylfamesyl diphosphate (FGPP); their monophosphorylated versionsgeranyl phosphate, farnesyl phosphate, geranylgeranyl phosphate, orgeranylfarnesyl phosphate; their alcohols geraniol, farnesol,geranylgeraniol, or geranylfarnesol; as well as downstream terpene andterpenoid products. MEP carbon further includes compounds derived fromFPP or pathways that use FPP, including squalene, undecaprenyldiphosphate (UPP), undecaprenyl phosphate, octaprenyl diphosphate (OPP),4-hydroxybenzoate, 3-octaprenyl-4-hydroxybenzoate, 2-octaprenylphenol,3-octaprenylbenzene-1,2-diol,2-methoxy-6-octaprenyl-2-methoxy-1,4-benzoquinol,6-methoxy-3-methyloctaprenyl-1,4-benzoquinol, 3-demethyluibquinol-8,ubiquinol-8, ubiquinone, 2-carboxy-1,4-naphthoquinol,demethylmenaquinol-8, menaquinol-8, and menaquinone. MEP carbon furtherincludes isoprenol, prenol, isopentenyl phosphate, and dimethylallylphosphate metabolites. MEP carbon (the intermediates and metabolitesabove) can be quantified by mass spectrometry (MS), such as tandem massspectrometry (MS/MS) via triple quadrupole (QQQ) mass detector. Anexemplary system is Agilent 6460 QQQ; alternatively, with quantitativetime-of-flight (QTOF), time-of-flight (TOF), or ion trap mass detectors.

In some embodiments, the microbial strain has at least one additionalcopy of dxs, ispD, ispF, and/or idi genes, which can be rate limiting,and which can be expressed from an operon or module, either on a plasmidor integrated into the bacterial chromosome. In some embodiments, thebacterial strain has at least one additional copy of dxs and idiexpressed as an operon/module; or dxs, ispD, ispF, and idi expressed asan operon or module. In some embodiments, the bacterial strain expressesdxs, dxr, ispD, ispE, ispF, and idi as recombinant genes, which areoptionally expressed as 1, 2, or 3 individual operons or modules. Therecombinant genes of the MEP pathway are expressed from one or moreplasmids or are integrated into the chromosome. In these embodiments,the strain provides increased flux through the MEP pathway as comparedto wild type.

Amino acid sequences for wild type E. coli enzymes Dxs, Dxr, IspD, IspE,IspF, and Idi are shown herein as SEQ ID NOS: 3 to 8. In variousembodiments, enzymes having structural or sequence homology, andcomparable functionality, can be employed (including bacterialhomologs). For example, the amino acid sequence may have 50% or moresequence identity with any one of SEQ ID NOS:3-8, or at least about 60%sequence identity, or at least about 70% sequence identity, or at leastabout 80% sequence identity, or at least about 90% sequence identity, orat least about 95% sequence identity, or at least about 98% sequenceidentity with the amino acid sequence of any one of SEQ ID NO:3-8. Insome embodiments, from 1 to about 10, or from 1 to about 5, amino acidsubstitutions, deletions, and/or insertions are made to the amino acidsequence (SEQ ID NO:3-8) to alter the activity of the protein, includingsubstitutions to one or more of the substrate binding site or activesite. Modifications to enzymes can be informed by construction of ahomology model. Such mutants can be informed by enzyme structuresavailable in the art, including Yajima S, et al., Structure of1-deoxy-D-xylulose 5-phosphate reductoisomerase in a quaternary complexwith a magnesium ion, NADPH and the antimalarial drug fosmidomycin, ActaCryst. F63, 466-470 (2007).

In some embodiments, the MEP complementation enhances conversion of DOXPand MEP pools to MEcPP, the substrate for IspG. See FIG. 1. Bottlenecksin the MEP pathway from dxs to ispF can be determined with regard toDOX, ME, and MEcPP levels, which can be detected extracellularly.Complementation and expression of MEP pathway enzymes can be balanced tomove carbon flux to MEcPP intermediate, as determined by metaboliteprofiling. In some embodiments, the expression or activity of IspG andIspH is balanced with respect to the expression or activity of Dxr, Dxs,IspD, IspE and IspF to pull MEcPP metabolite to IPP and DMAPPprecursors. MEcPP can be transported to the extracellular medium, andthus large MEcPP pools can result in lost MEP carbon.

In some embodiments, the expression or activity of a recombinant idigene is tuned to increase terpene or terpenoid production. The Idienzyme catalyzes the reversible isomerization of IPP to DMAPP. Sinceevery desired terpenoid product or undesired MEP side-product (e.g.,UPP) uses one DMAPP and varying numbers of IPP, the ratio between thetwo precursors can have an impact on strain productivity. Varying theratio of IPP:DMAPP available, e.g., by varying Idi expression oractivity, can have an impact on the production of the desired terpenoidrelative to other undesired products from the MEP pathway. For example,as shown in FIG. 9, while Idi overexpression slightly increases producttiter in a strain that does not overexpress IspGH (Strain 1), itdecreases titer in two strains that do (Strains 2 and 3), indicatingthat the balance between IPP and DMAPP controlled by Idi can be tuned upor down depending on the needs of the downstream pathway. However,Strain 4 (FIG. 9), which has a different balance of MEP pathway enzymeexpression, more than doubles titer with Idi complementation. Theexpression of the recombinant idi gene can be tuned in variousembodiments by modifying the promoter strength, gene copy number,position in an operon, or ribosome binding site, in addition to pointmutations to increase or decrease enzyme productivity.

The microbial strain provides substantial increases in MEP carbon,including substantial increases in IPP and DMAPP precursor flux, withoutsubstantial impact on strain growth and viability, for example, asdetermined by optical density (O.D.) in culture, peak O.D., and/orgrowth rate. For example, despite increased flux through the MEPpathway, which is tightly controlled in bacterial cells, the microbialstrain does not have a drop in peak O.D. of more than about 20%, or insome embodiments, does not have a drop in peak O.D. of more than about15%, or more than about 10%, or more than about 5%. In some embodiments,the strain does not exhibit a measurable impact on strain growth orviability, as determined for example by measuring growth rate or peakO.D.

In some embodiments, the bacterial strain contains one or more geneticmodifications that enhance the supply and transfer of electrons throughthe MEP pathway, and/or to terpene or terpenoid products. In someembodiments, the enhanced supply and transfer of electrons through theMEP pathway is by recombinant expression of one or more oxidoreductaseenzymes, including oxidoreductases that oxidize pyruvate and/or lead toreduction of ferredoxin. Ferredoxin supplies electrons to the MEPpathway and supports activity of IspG and IspH (which are Fe—S clusterenzymes). See FIG. 10. In various embodiments, the microbial straincomprises an overexpression of or complementation with one or more of aflavodoxin (fldA), flavodoxin reductase, ferredoxin (fdx), andferredoxin reductase.

By way of example, in some embodiments, the oxidoreductase is apyruvate:flavodoxin oxidoreductase (PFOR). In some embodiments, the PFORis YdbK. In some embodiments, the YdbK is E. coli YdbK, or orthologs andderivatives thereof.

In some embodiments, the strain contains a complementation oroverexpression of YdbK. YdbK is predicted to function as apyruvate:flavodoxin oxidoreductase and/or pyruvate synthase. Theoxidoreductase is thought to oxidize pyruvate to acetyl-CoA, reducingferredoxin, which can then supply electrons to the MEP pathway,especially to support the strongly upregulated IspG and IspH enzymesthat contain Fe—S clusters. In some embodiments, the expression of arecombinant YdbK is balanced with the expression of IspG and IspH, whichcan be determined by product titer (or farnesol titer as describedbelow). In some embodiments, the YdbK gene is under the control of aweak or intermediate strength promoter. Additionally, extraelectron-carrying or transferring cofactors can be expressed on top ofYdbK overexpression. See, e.g., Akhtar, et al., Metabolic Engineering,11(3): 139-147 (2009). In some experiments, YdbK is overexpressed withfdx (ferredoxin) from Clostridium pasteurianum (SEQ ID NO:10) and/or E.Coli (Ec.ydhY) (SEQ ID NO: 34), or enzyme having at least 80% or atleast 90% sequence identity therewith. The bacterial strain may comprisea recombinant YdbK gene, which may be integrated into the chromosome orexpressed from a plasmid. The amino acid sequence of the E. coli YdbKenzyme is shown herein as SEQ ID NO:9. In various embodiments, enzymeshaving structural or sequence homology, and comparable functionality,can be employed. For example, the amino acid sequence may have 50% ormore sequence identity with any one of SEQ ID NO:9, or at least about60% sequence identity, or at least about 70% sequence identity, or atleast about 80% sequence identity, or at least about 90% sequenceidentity, or at least about 95% sequence identity, or at least about 97%sequence identity, or at least about 98% sequence identity with theamino acid sequence of SEQ ID NO:9. In some embodiments, from 1 to about10, or from 1 to about 5 amino acid substitutions, deletions, and/orinsertions are made to the amino acid sequence (SEQ ID NO:9) to alterthe activity of the protein.

In some embodiments, the strain comprises one or more P450 enzymes forthe production of a terpenoid compound. The overexpression of YdbK andpotentially other oxidoreductases, might support higher levels of P450oxidative chemistry.

In some embodiments, including in embodiments where the bacterial strainoverexpresses or has higher activity of pyruvate:flavodoxinoxidoreductase (PFOR), the strain exhibits reduced conversion ofpyruvate to acetyl-COA by pyruvate dehydrogenase (PDH). In someembodiments, the conversion of pyruvate to acetyl-COA by PDH is reducedby deleting or inactivating PDH, or by reducing expression or activityof PDH. In some embodiments, PDH is deleted. Alternatively, activity ofPDH may be reduced by one or more amino acid modifications. An exemplarymutation to reduce PDH activity is a G267C mutation in aceE.

In some embodiments, the conversion of pyruvate to acetyl-COA by PDH isreduced by modifying the aceE-aceF-lpd complex of PDH. In someembodiments, the aceE-aceF-lpd complex is modified by the deletion,inactivation, or reduced expression or activity of aceE, aceF, lpd, or acombination thereof. By way of example, in some embodiments, aceE isdeleted (e.g., by knockout). Alternatively, in some embodiments, theaceE-aceF-pd complex is modified by one or more mutations of aceE, aceF,lpd, or a combination thereof.

By reducing conversion of pyruvate to acetyl-COA by PDH, the bacterialstrain will rely more on PFOR (e.g., YdbK) for the conversion ofpyruvate to acetyl-COA. See FIG. 15. This reliance enhances IspG andIspH activity.

In some embodiments, supply and transfer of electrons to IspG and IspHis improved by overexpression or complementation with one or moreoxidoreductases, such as, e.g., PFOR. By way of example, in someembodiments, the PFOR, or a homolog thereof, is selected from YdbK (SEQID NO: 9), Scy.pfor (Synechocystis sp.) (SEQ ID NO: 29), Ki.pfor(Kluyvera intermedia) (SEQ ID NO: 30), Da.pfor (Desulfovibrio africanus)(SEQ ID NO: 31), Ns.pfor (Nostoc sp.) (SEQ ID NO: 32), Ec.ydhV (E. Coli)(SEQ ID NO: 33), Ga.pfor (Gilliamella apicola) (SEQ ID NO: 35), andSco.pfor (Synechococcus sp.). In some embodiments, the PFOR is YdbK.

In some embodiments, the PFOR comprise a sequence that is at least 60%identical to any one of SEQ ID NOs. 29-35. For example, the PFOR cancomprise a sequence that is at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about97%, at least about 98%, at least about 99%, or 100% identical to any ofone of SEQ ID NOs. 29-35.

In some embodiments, the overexpression or complementation with PFORsuch as, e.g., YdbK, can result in improved performance throughexpression of electron carriers having a redox potential of about 400 to550 mV, or in some embodiments, in the range of about 400 to 500 mV, orin the range of about 400 to 475 mV. In some embodiment, the electroncarrier is ferrodoxin, flavodoxin, or NADPH. By way of example, in someembodiments, the electron carrier is Cv.fdx (Allochromatium vinosum).

In some embodiments, the bacterial strain has overexpression orcomplementation with one or more fpr homologs. By way of example, insome embodiments, the fpr homolog is selected from Ns.fpr (Nostoc sp.)(SEQ ID NO: 36), Sco.fpr (Synechococcus sp.) (SEQ ID NO: 37), and Ec.fpr(E. Coli) (SEQ ID NO: 38).

In some embodiments, the fpr comprise a sequence that is at least 60%identical to any one of SEQ ID NOs. 36-38. For example, the fpr cancomprise a sequence that is at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about97%, at least about 98%, at least about 99%, or 100% identical to any ofone of SEQ ID NOs: 36-38.

In some embodiments, the bacterial strain overexpressing YdbK or homologor derivative thereof, further expresses a non-native electronacceptor/donor, such as one or more non-native fdx and/or fldA homologs.By way of example, the fdx homolog may be selected from Hm.fdx1(Heliobacterium modesticaldum) (SEQ ID NO: 15), Pa.fdx (Pseudomonasaeruginosa) (SEQ ID NO: 16), Cv.fdx (Allochromatium vinosum) (SEQ ID NO:17), Cv.fdx_C57A (synthetic) (SEQ ID NO: 18), Ec.yfhL (E. Coli) (SEQ IDNO: 19), Ca.fdx (Clostridium acetobutylicum) (SEQ ID NO: 20), Cp.fdx(Clostridium pasteurianum) (SEQ ID NO: 10), Ec.fdx (E. Coli) (SEQ ID NO:21), Ev2.fdx (Ectothiorhodospira shaposhnikovii) (SEQ ID NO: 22),Ppl.fdx (Pseudomonas putida) (SEQ ID NO: 23), and Pp2.fdx (Pseudomonasputida) (SEQ ID NO: 24). In some embodiments, the fldA homolog includesone or more selected from Ec.fldA (E. coli) (SEQ ID NO: 27), Ac.fldA2(Azotobacter chroococcum) (SEQ ID NO: 26), Av.fldA2 (Azotobactervinelandii) (SEQ ID NO: 25), and Bs.fldA (B. subtilis) (SEQ ID NO: 28).Expression of a non-native fdx homolog and/or fldA homolog results in anincreased supply of electrons to IspG and/or IspH, an increase in IspG/Hactivity, and an increase in terpenoid production. See FIGS. 19A-C.

In some embodiments, the non-native fdx homologs comprise a sequencethat is at least 60% identical to any one of SEQ ID NOs. 10 and 15-24.For example, the non-native fdx homologs can comprise a sequence that isat least about 60%, at least about 70%, at least about 80%, at leastabout 90°, at least about 95%, at least about 97%, at least about 98%,at least about 99%, or 100% identical to any of one of SEQ ID NOs. 10and 15-24.

In some embodiments, the non-native fldA homologs comprise a sequencethat is at least 60% identical to any one of SEQ ID NOs. 25-28. Forexample, the non-native fldA homologs can comprise a sequence that is atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 97%, at least about 98%, atleast about 99%, or 100% identical to any of one of SEQ ID NOs. 25-28.

In some embodiments, the bacterial strain has overexpression orcomplementation with one or more PFOR and/or fpr and, optionally, one ormore of a flavodoxin (fldA), flavodoxin reductase, ferredoxin (fdx), andferredoxin reductase. By way of example, in some embodiments thebacterial strain includes Ec.ydhV (E. Coli) (SEQ ID NO: 33) and Ec.ydhY(E Coli) (SEQ ID NO: 34); Ec.ydbK (E Coli) (SEQ ID NO: 9) and Cp.fdx(Clostridium pasteurianum) (SEQ ID NO: 10); Ec.fpr (E. Coli) (SEQ ID NO:38) and Ec.fdx (E Coli) (SEQ ID NO: 21); or Ec.fpr (E Coli) (SEQ ID NO:38) and Ec.fldA (E coli) (SEQ ID NO: 27).

In other aspects, the invention provides bacterial strains thatoverexpress PgpB or NudB enzymes, for increasing MEP carbon pull.Installing this alternate ‘product’ pull by overexpressing genes such aspgpB and mdB pulls even more flux through the MEP pathway (though tonon-target products) and minimizes the accumulation of potentially toxicor feedback inhibitory intermediates (e.g., IPP, DMAPP, FPP). In someembodiments, the PgpB or NudB overexpression is in the absence of adownstream terpenoid pathway, thereby creating a ‘universal chassis’;that is, a strain that can have any terpenoid downstream transformedinto it and be quickly optimized for commercial production.

More specifically, carbon can be pulled through the MEP pathway tocreate alternate products that will pool outside the cell. PgpBdephosphorylates FPP to farnesol (FOH), and NudB dephosphorylates IPPand DMAPP to isoprenol (3-methyl-3-buten-1-ol) and prenol(3-methyl-2-buten-1-ol), respectively (See FIG. 1). Enhancing transportof these products outside the cell prevents buildup of IPP, DMAPP, andFPP; which like HMBPP, can feedback and exert control on the MEPpathway. IPP inhibits growth and feedback inhibits Dxs. See Cordoba,Salmi & Leon (2009) J. Exp. Bot. 60, 10, 2933-2943. FPP inhibits growthand feedback inhibits ispF-MEP complex, which itself is formed when MEPbinds and enhances IspF activity in a feed-forward manner. Bitok &Meyers (2012) ACS Chem. Biol. 2012, 7, 1702-1710. Farnesol, isoprenol,and prenol accumulate outside the cell and, like the intermediates inthe MEP pathway, can be used to track carbon flux through the MEPpathway via LC/MS or GC/MS quantitation.

In various embodiments, enzymes having structural or sequence homology,and comparable functionality, can be employed. For example, the aminoacid sequence may have 50% or more sequence identity with either SEQ IDNOS:11 (PgpB) or 12 (NudB), or at least about 60% sequence identity, orat least about 70% sequence identity, or at least about 80% sequenceidentity, or at least about 90% sequence identity, or at least about 95%sequence identity with the amino acid sequence of SEQ ID NO:11 or 12. Insome embodiments, from 1 to about 10, or from 1 to about 5 amino acidsubstitutions, deletions, and/or insertions are made to the amino acidsequence (SEQ ID NO:11 or 12) to alter the activity of the protein,including substitutions to one or more of the substrate binding site oractive site.

Thus, by constitutively expressing an additional copy of pgpB or nudB,carbon flux through the MEP pathway can be improved, and a slow growthphenotype ameliorated. In cases where ispG and ispH are balanced andpgpB or nudB are overexpressed, the increase or decrease in farnesolproduct is inversely correlated with MEcPP level (FIGS. 7 and 8).

However, too much PgpB or NudB expression might negatively impact thetotal flux through to farnesol, with lower titer and smallerfold-change. See FIG. 6. Thus, in various embodiments, the expression ofthe recombinant PgpB and/or NudB is tuned to provide higher terpene orterpenoid product titer, optionally by varying promoter strength, genecopy number, position in an operon, and/or ribosome binding site. Insome embodiments, the recombinant pgpB and/or nudB genes are expressedunder control of a weak or intermediate strength promoter. Therecombinant pgpB or nudB can be integrated into the chromosome orexpressed from a plasmid.

In some embodiments, the bacterial strains overexpress one or moresynthases for increasing MEP carbon pull. By way of example, in someembodiments, the synthase is selected from Artemisia annua farnesenesynthase and valencene synthase.

In some embodiments, the bacterial strain has one or more additionalmodifications to increase co-factor availability or turnover, includingNADH and NADPH cofactor, thereby leading to increases in MEP carbon. SeeFIG. 14. In some embodiments, the bacterial strain expresses aglyceraldehyde 3-phosphate ferredoxin oxidoreductase (GAPOR), forexample, from Methanococcus maripaludis (SEQ ID NO: 14) or othermesophilic organism. Expression of a GAPOR would provide electrons toferredoxins in central carbon metabolism, and could provide electronsfor IspG, IspH or P450 enzymes. In some embodiments, the bacterialstrain overexpresses one or more genes of the ydh operon, such as ydhVor ydhY (e.g., by complementing the wild-type gene or enhancingexpression of the endogenous bacterial gene). YdhV or other bacterialgenes having GAPOR-like activity can increase co-factor availability tofurther enhance MEP carbon. Other genetic modifications includedownregulation, inactivation, or deletion of gshA or expression of CHAC1and/or CHAC2 (e.g., from Homo sapiens). These modifications can alterglutathione levels, thereby indirectly increasing NADPH availability.

While various bacterial species can be modified in accordance with thedisclosure, in some embodiments, the bacterial strain is a bacteriaselected from Escherichia spp., Bacillus spp., Corynebacterium spp.,Rhodobacter spp., Zymomonas spp., Vibrio spp., and Pseudomonas spp. Insome embodiments, the bacterial strain is a species selected fromEscherichia coli, Bacillus subtilis, Corynebacterium glutamicum,Rhodobacter capsulatus, Rhodobacter sphaeroides, Zymomonas mobilis,Vibrio natriegens, or Pseudomonas putida. In some embodiments, thebacterial strain is E. coli.

In accordance with embodiments described herein, various strategies canbe employed for engineering the expression or activity of recombinantgenes and enzymes, including, for example, modifications or replacementof promoters of different strengths, modifications to the ribosomebinding sequence, modifications to the order of genes in an operon ormodule, gene codon usage, RNA or protein stability, RNA secondarystructure, and gene copy number, among others.

In some embodiments, the ribosome binding site sequence can be altered,to tune translation of the mRNA. The Shine-Dalgarno (SD) sequence is theribosomal binding site in bacteria and is generally located around 8bases upstream of the start codon AUG. The RNA sequence helps recruitthe ribosome to the messenger RNA (mRNA) to initiate protein synthesisby aligning the ribosome with the start codon. The six-base consensussequence is AGGAGG (SEQ ID NO:13) in Escherichia coli. Mutations in theconsensus sequence can be screened for improvements in product titer(including farnesol titer in some embodiments), or screened bymetabolomic analysis of MEP carbon.

For complementation of genes, wild type genes can be employed, and insome embodiments, the gene is a wild-type E. coli gene. Alternatively,various orthologs can be employed, which may show nucleotide or aminoacid homology to the E. coli gene. Exemplary genes can be derived fromthe orthologs of Bacillus spp., Corynebacterium spp., Rhodobacter spp.,Zymomonas spp., Vibrio spp., Pseudomonas spp., Chloroboculum spp.,Synechocystis sp., Burkholderia spp., and Stevia rebaudiana, forexample.

The similarity of nucleotide and amino acid sequences, i.e. thepercentage of sequence identity, can be determined via sequencealignments. Such alignments can be carried out with several art-knownalgorithms, such as with the mathematical algorithm of Karlin andAltschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) orwith the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T.J. (1994) Nucleic Acids Res. 22, 4673-80). The grade of sequenceidentity (sequence matching) may be calculated using e.g. BLAST, BLAT orBlastZ (or BlastX). A similar algorithm is incorporated into the BLASTNand BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215: 403-410.BLAST polynucleotide searches can be performed with the BLASTN program,score=100, word length=12.

BLAST protein searches may be performed with the BLASTP program,score=50, word length=3. To obtain gapped alignments for comparativepurposes, Gapped BLAST is utilized as described in Altschul et al (1997)Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs are used.Sequence matching analysis may be supplemented by established homologymapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b,19 Suppl 1:154-162) or Markov random fields.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups:

-   -   (1) hydrophobic: Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro; and    -   (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt α-helices. Some preferred conservativesubstitutions within the above six groups are exchanges within thefollowing sub-groups: (i) Ala, Val, Leu and Ile; (ii) Ser and Thr; (ii)Asn and Gin; (iv) Lys and Arg; and (v) Tyr and Phe.

As used herein, “non-conservative substitutions” are defined asexchanges of an amino acid by another amino acid listed in a differentgroup of the six standard amino acid groups (1) to (6) shown above.

Modifications of enzymes as described herein can include conservativeand/or non-conservative mutations.

In some embodiments “rational design” is involved in constructingspecific mutations in enzymes. Rational design refers to incorporatingknowledge of the enzyme, or related enzymes, such as its reactionthermodynamics and kinetics, its three dimensional structure, its activesite(s), its substrate(s) and/or the interaction between the enzyme andsubstrate, into the design of the specific mutation. Based on a rationaldesign approach, mutations can be created in an enzyme which can then bescreened for increased production of a terpene or terpenoid relative toparent strain levels, or metabolite profile that corresponds withimprovements in MEP carbon. In some embodiments, mutations can berationally designed based on homology modeling. “Homology modeling”refers to the process of constructing an atomic resolution model of aprotein from its amino acid sequence, using the three-dimensionalstructure of a related homologous protein.

Amino acid modifications can be made to enzymes to increase or decreaseactivity of the enzyme or enzyme complex. Gene mutations can beperformed using any genetic mutation method known in the art. In someembodiment, a gene knockout eliminates a gene product in whole or inpart. Gene knockouts can be performed using any knockout method known inthe art.

Manipulation of the expression of genes and/or proteins, including genemodules, can be achieved through various methods. For example,expression of the genes or operons can be regulated through selection ofpromoters, such as inducible or constitutive promoters, with differentstrengths (e.g., strong, intermediate, or weak). Several non-limitingexamples of promoters include Trc, T5 and T7. Additionally, expressionof genes or operons can be regulated through manipulation of the copynumber of the gene or operon in the cell. In some embodiments,expression of genes or operons can be regulated through manipulating theorder of the genes within a module, where the genes transcribed firstare generally expressed at a higher level. In some embodiments,expression of genes or operons is regulated through integration of oneor more genes or operons into the chromosome.

In some embodiments, balancing gene expression includes the selection ofhigh-copy number plasmids, or single-, low- or medium-copy numberplasmids. In still other embodiments, the step of transcriptiontermination can also be targeted for regulation of gene expression,through the introduction or elimination of structures such asstem-loops.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA. The heterologous DNA is placed under operable controlof transcriptional elements to permit the expression of the heterologousDNA in the host cell.

In some embodiments, endogenous genes are edited, as opposed to genecomplementation. Editing can modify endogenous promoters, ribosomalbinding sequences, or other expression control sequences, and/or in someembodiments modifies trans-acting and/or cis-acting factors in generegulation. Genome editing can take place using CRISPR/Cas genomeediting techniques, or similar techniques employing zinc fingernucleases and TALENs. In some embodiments, the endogenous genes arereplaced by homologous recombination.

In some embodiments, genes are overexpressed at least in part bycontrolling gene copy number. While gene copy number can be convenientlycontrolled using plasmids with varying copy number, gene duplication andchromosomal integration can also be employed. For example, a process forgenetically stable tandem gene duplication is described in US2011/0236927, which is hereby incorporated by reference in its entirety.

In certain embodiments, the bacterial cell produces one or more terpeneor terpenoid compounds. A terpenoid, also referred to as an isoprenoid,is an organic chemical derived from a five-carbon isoprene unit (C).Several non-limiting examples of terpenoids, classified based on thenumber of isoprene units that they contain, include: hemiterpenoids (1isoprene unit), monoterpenoids (2 isoprene units), sesquiterpenoids (3isoprene units), diterpenoids (4 isoprene units), sesterterpenoids (5isoprene units), triterpenoids (6 isoprene units), tetraterpenoids (8isoprene units), and polyterpenoids with a larger number of isopreneunits. In an embodiment, the bacterial host cell produces a terpenoidselected from a monoterpenoid, a sesquiterpenoid, diterpenoid, asesterpenoid, or a triterpenoid. Terpenoids represent a diverse class ofmolecules that provide numerous commercial applications, including inthe food and beverage industries as well as the perfume, cosmetic andhealth care industries. By way of example, terpenoid compounds find usein perfumery (e.g. patchoulol), in the flavor industry (e.g.,nootkatone), as sweeteners (e.g., steviol), colorants, or therapeuticagents (e.g., taxol) and many are conventionally extracted from plants.Nevertheless, terpenoid molecules are found in ppm levels in nature, andtherefore require massive harvesting to obtain sufficient amounts forcommercial applications.

The bacterial cell will generally contain a recombinant downstreampathway that produces the terpenoid from IPP and DMAPP precursors.Terpenes such as Monoterpenes (C10), Sesquiterpenes (C15), Diterpenes(C20), Sesterterpenes (C25), and Triterpenes (C30) are derived from theprenyl diphosphate substrates, geranyl diphosphate (GPP), farnesyldiphosphate (FPP) geranylgeranyl diphosphate (GGPP), geranylfarnesyldiphosphate (FGPP), and two FPP, respectively, through the action of avery large group of enzymes called the terpene (terpenoid) synthases.These enzymes are often referred to as terpene cyclases since theproduct of the reactions are cyclized to various monoterpene,sesquiterpene, diterpene, sesterterpene and triterpene carbon skeletonproducts. Many of the resulting carbon skeletons undergo subsequenceoxygenation by cytochrome P450 enzymes to give rise to large families ofderivatives.

Exemplary terpene or terpenoid products that may be produced inaccordance with the invention are described in U.S. Pat. No. 8,927,241,which is hereby incorporated by reference, and include: farnesene,amorphadiene, artemisinic acid, artemisinin, bisabolol, bisabolene,alpha-Sinensal, beta-Thujone, Camphor, Carveol, Carvone, Cineole,Citral, Citronellal, Cubebol, Geraniol, Limonene, Menthol, Menthone,Myrcene, Nootkatone, Nootkatol, Patchouli, Piperitone, Rose oxide,Sabinene, Steviol, Steviol glycoside (including Rebaudioside D orRebaudioside M), Taxadiene, Thymol, and Valencene. Enzymes forrecombinantly constructing the pathways in E. coli are described in U.S.Pat. No. 8,927,241, WO 2016/073740, and WO 2016/029153, which are herebyincorporated by reference.

Exemplary P450 enzymes that are operative on sesquiterpene scaffolds toproduce oxygenated terpenoids are described in WO 2016/029153, which ishereby incorporated by reference. In addition, P450 reductase proteinsthat find use in the bacterial strains described herein are described inWO 2016/029153 as well as WO 2016/073740.

As used herein, the term “oxygenated terpenoid” refers to a terpenescaffold having one or more oxygenation events, producing acorresponding alcohol, aldehyde, carboxylic acid and/or ketone. In someembodiments, the bacterial cell produces at least one terpenoid selectedfrom Abietadiene, Abietic Acid, alpha-Sinensal, beta-Thujone, Camphor,Carveol, Carvone, Celastrol, Ceroplastol, Cineole, Citral, Citronellal,Cubebol, Cucurbitane, Forskolin, Gascardic Acid, Geraniol, Haslene,Levopimaric Acid, Limonene, Lupeol, Menthol, Menthone, Mogroside,Nootkatone, Nootkatol, Ophiobolin A, Patchouli, Piperitone, RebaudiosideD, Rebaudioside M, Sabinene, Steviol, Steviol glycoside, Taxadiene,Thymol, and Ursolic Acid.

In some embodiments, the terpenoid synthase enzyme is upgraded toenhance the kinetics, stability, product profile, and/or temperaturetolerance of the enzyme, as disclosed, for example, in WO 2016/029153and WO 2016/073740, which are hereby incorporated by reference.

In another embodiment, the bacterial cell produces valencene and/ornootkatone. In such an embodiment, the bacterial cell may express abiosynthetic pathway that further includes a farnesyl diphosphatesynthase, a Valencene Synthase, and a Valencene Oxidase. Farnesyldiphosphate synthases (FPPS) produce famesyl diphosphates fromisopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Anexemplary famesyl diphosphate synthase is ERG20 of Saccharomycescerevisiae (NCBI accession P08524) and E. coli ispA. Valencene synthaseproduces sesquiterpene scaffolds and are described in, for example, US2012/0107893, US 2012/0246767, and U.S. Pat. No. 7,273,735, which arehereby incorporated by reference in their entireties. Genes and hostcells for the production of terpenoid product comprising valenceneand/or nootkatone are described in WO 2016/029153, which is herebyincorporated by reference.

In an embodiment, the bacterial cell produces steviol or steviolglycoside (e.g., RebD or RebM). Steviol is produced from kaurene by theaction of two P450 enzymes, kaurene oxidase (KO) and kaurenoic acidhydroxylase (KAH). After production of steviol, various steviolglycoside products may be produced through a series of glycosylationreactions, which can take place in vitro or in vivo. Pathways andenzymes for production of steviol and steviol glycosides are disclosedin US 2013/0171328, US 2012/0107893, WO 2012/075030, WO 2014/122328,which are hereby incorporated by reference in their entireties. WO2016/073740 further discloses enzymes and bacterial host cells forproduction of RebM.

Other biosynthetic pathways for production of terpene or terpenoidcompounds are disclosed in U.S. Pat. No. 8,927,241, which is herebyincorporated by reference in its entirety.

The bacterial strain may be cultured in batch culture, continuousculture, or semi-continuous culture. In some embodiments, the bacterialstrain is cultured using a fed-batch process comprising a first phasewhere bacterial biomass is created, followed by a terpene or terpenoidproduction phase. Fed-batch culture is a process where nutrients are fedto the bioreactor during cultivation and in which the product(s) remainin the bioreactor until the end of the run. Generally, a base mediumsupports initial cell culture and a feed medium is added to preventnutrient depletion. The controlled addition of the nutrient directlyaffects the growth rate of the culture and helps to avoid overflowmetabolism and formation of side metabolites.

An exemplary batch media for growing the bacterial strain (producingbiomass) comprises, without limitation, yeast extract. In someembodiments, carbon substrates such C1, C2, C3, C4, C5, and/or C6 carbonsubstrates are fed to the culture for production of the terpene orterpenoid product. In exemplary embodiments, the carbon source isglucose, sucrose, fructose, xylose, and/or glycerol. Culture conditionsare generally selected from aerobic, microaerobic, and anaerobic.

In some embodiments, the culture is maintained under aerobic conditions,or microaerobic conditions. For example, when using a fed-batch process,the biomass production phase can take place under aerobic conditions,followed by reducing the oxygen levels for the product production phase.For example, the culture can be shifted to microaerobic conditions afterfrom about 10 to about 20 hours. In this context, the term “microaerobicconditions” means that cultures are maintained just below detectabledissolved oxygen. See, Partridge J D, et al., Transition of Escherichiacoli from Aerobic to Micro-aerobic Conditions Involves Fast and SlowReacting Regulatory Components, J. Biol. Chem. 282(15):11230-11237(2007).

The production phase includes feeding a nitrogen source and a carbonsource. For example, the nitrogen source can comprise ammonium (e.g.,ammonium hydroxide). The carbon source may contain C1, C2, C3, C4, C5,and/or C6 carbon sources, such as, in some embodiments, glucose,sucrose, or glycerol. The nitrogen and carbon feeding can be initiatedwhen a predetermined amount of batch media is consumed, a process thatprovides for ease of scaling. In some embodiments, the nitrogen feedrate is from about 8 L per hour to about 20 L per hour, but will dependin-part on the product, strain, and scale.

In various embodiments, the bacterial host cell may be cultured at atemperature between 22° C. and 37° C. While commercial biosynthesis inbacteria such as E. coli can be limited by the temperature at whichoverexpressed and/or foreign enzymes are stable, recombinant enzymes(including the terpenoid synthase) may be engineered to allow forcultures to be maintained at higher temperatures, resulting in higheryields and higher overall productivity. In some embodiments, theculturing is conducted at about 22° C. or greater, about 23° C. orgreater, about 24° C. or greater, about 25° C. or greater, about 26° C.or greater, about 27° C. or greater, about 28° C. or greater, about 29°C. or greater, about 30° C. or greater, about 31° C. or greater, about32° C. or greater, about 33° C. or greater, about 34° C. or greater,about 35° C. or greater, about 36° C. or greater, or about 37° C. Insome embodiments, the culture is maintained at a temperature of from 22to 37° C., or a temperature of from 25 to 37° C., or a temperature offrom 27 to 37° C., or a temperature of from 30 to 37° C.

In some embodiments, the bacterial strain is cultured at commercialscale. In some embodiments, the size of the culture is at least about100 L, or at least about 200 L, or at least about 500 L, or at leastabout 1,000 L, or at least about 10,000 L, or at least about 100,000 L,or at least about 500,000 L. In some embodiments, the culture is fromabout 300 L to about 1,000,000 L.

In various embodiments, methods further include recovering the terpeneor terpenoid product from the cell culture or from cell lysates. In someembodiments, the culture produces at least about 100 mg/L, at leastabout 150 mg/L, or at least about 200 mg/L, or at least about 500 mg/L,or at least about 1 g/L, or at least about 5 g/L, or at least about 10g/L, or at least about 15 g/L of the terpene or terpenoid product.

In some embodiments, the production of indole is used as a surrogatemarker for terpenoid production, and/or the accumulation of indole inthe culture is controlled to increase production. For example, invarious embodiments, accumulation of indole in the culture is controlledto below about 100 mg/L, or below about 75 mg/L, or below about 50 mg/L,or below about 25 mg/L, or below about 10 mg/L. The accumulation ofindole can be controlled by balancing enzyme expression (and inparticular, balancing the upstream and downstream pathways) and activityusing the multivariate modular approach as described in U.S. Pat. No.8,927,241 (which is hereby incorporated by reference). In someembodiments, the accumulation of indole is controlled by chemical means.

Other markers for efficient production of terpene and terpenoids,include accumulation of DOX or ME in the culture media. Generally, thebacterial strains described herein do not accumulate large amounts ofthese chemical species, which accumulate in the culture at less thanabout 5 g/L, or less than about 4 g/L, or less than about 3 g/L, or lessthan about 2 g/L, or less than about 1 g/L, or less than about 500 mg/L,or less than about 100 mg/L.

In some embodiments, MEcPP is the predominant MEP metabolite in theculture media, although its accumulation is limited by the geneticmodifications to the bacterial strain, which pull MEP carbon downstreamto IPP and DMAPP precursors. In various embodiments, MEcPP accumulatesin the culture at less than about 30 g/L, or less than about 20 g/L, orless than about 2 g/L, or less than about 1 g/L, or less than about 500mg/L, or less than about 100 mg/L.

The optimization of terpene or terpenoid production by manipulation ofMEP pathway genes, as well as manipulation of the upstream anddownstream pathways, is not expected to be a simple linear or additiveprocess. Rather, through combinatorial analysis, optimization isachieved through balancing components of the MEP pathway, as well asupstream and downstream pathways. Indole accumulation (includingprenylated indole) and MEP metabolite accumulation (e.g., DOX, ME,MEcPP, HMBPP, farnesol, prenol and isoprenol) in the culture or cellscan be used as surrogate markers to guide this process.

The terpene or terpenoid product can be recovered by any suitableprocess. Generally, recovery includes separation of material comprisingproduct from the culture or cells, followed by extraction andpurification. For example recovery can include partitioning the desiredproduct into an organic phase or hydrophobic phase. Alternatively, theaqueous phase can be recovered, or the whole cell biomass can berecovered, for further processing.

For example, in some embodiments, the product is a volatile terpene orterpenoid product. In such embodiments, the terpene or terpenoid productcan be recovered from an organic or hydrophobic phase that ismechanically separated from the culture. Alternatively or in addition,the terpene or terpenoid product is harvested from the liquid and/orsolid phase. In some embodiments, the product is purified by sequentialextraction and purification. For example, the product may be purified bychromatography-based separation and recovery, such as supercriticalfluid chromatography. The product may be purified by distillation,including simple distillation, steam distillation, fractionaldistillation, wipe-film distillation, or continuous distillation.

In some embodiments, the product is a non-volatile terpene or terpenoidproduct, which in some embodiments is an extracellular product recoveredfrom the culture medium. Alternatively, the product is an intracellularproduct recovered from harvested cell material. Where the product ispoorly soluble, it may be recovered by filtration, and optionally withsolvent extraction (e.g., extraction with ethanol). Alternatively, or inaddition, the product is recovered by chromatography-based separation,such as liquid chromatography. In some embodiments, the product isrecovered by sequential extraction and purification. In still otherembodiments, the product is crystallized out of solution.

The production of the desired product can be determined and/orquantified, for example, by gas chromatography (e.g., GC-MS). Productionof product, recovery, and/or analysis of the product can be done asdescribed in US 2012/0246767, which is hereby incorporated by referencein its entirety. For example, in some embodiments, product oil isextracted from aqueous reaction medium using an organic solvent, such asan alkane such as heptane or dodecane, followed by fractionaldistillation. In other embodiments, product oil is extracted fromaqueous reaction medium using a hydrophobic phase, such as a vegetableoil, followed by organic solvent extraction and fractional distillation.Terpene and terpenoid components of fractions may be measuredquantitatively by GC/MS, followed by blending of fractions to generate adesired product profile.

In various embodiments, the recovered terpene or terpenoid isincorporated into a product (e.g., a consumer or industrial product).For example, the product may be a flavor product, a fragrance product, asweetener, a cosmetic, a cleaning product, a detergent or soap, or apest control product. For example, in some embodiments, the productrecovered comprises nootkatone, and the product is a flavor productselected from a beverage, a chewing gum, a candy, or a flavor additive,or the product is an insect repellant. In some embodiments, theoxygenated product is steviol or a steviol glycoside (e.g., RebM), whichis provided as a sweetener, or is incorporated into ingredients,flavors, beverages or food products.

The invention further provides methods of making products such as foods,beverages, texturants (e.g., starches, fibers, gums, fats and fatmimetics, and emulsifiers), pharmaceutical products, tobacco products,nutraceutical products, oral hygiene products, and cosmetic products, byincorporating the terpene or terpenoids produced herein. The higheryields of such species produced in embodiments of the invention canprovide significant cost advantages as well as sustainability.

In other aspects, the invention provides bacterial cells, such as E.coli, having one or more genetic modifications that increase products ofIPP and DMAPP precursors. In various embodiments, the bacterial cellsproduce isopentenyl diphosphate (IPP) and dimethylallyl diphosphate(DMAPP) through the MEP pathway, and convert the IPP and DMAPP to aterpene or terpenoid product through a downstream synthesis pathway. Thedownstream synthesis pathway is generally a recombinant pathway, and maycomprise a prenyl transferase, one or more terpene synthases, andoptionally one or more P450 enzymes and P450 reductase enzymes (forexample, each as described above). For example, the product may be aditerpene or diterpenoid, with the sequential action of a recombinantType II diterpene synthase (DiTPS) on GGPP followed by a recombinantType I DiTPS, or alternatively, a single recombinant synthase performsboth steps.

Further, to improve MEP carbon available for product biosynthesis, thebacterial strain has one or more of the following genetic modifications:

(a) overexpression of IspG and IspH enzymes, the IspG and IspH enzymeshaving balanced expression to prevent accumulation of HMBPPintermediate,

(b) a recombinant or modified gene encoding an enzyme that enhancessupply and/or transfer of electrons through the MEP pathway and/or toterpene or terpenoid products, which is optionally an overexpression ofa YdbK gene and optionally with a non-native fdx and/or fldA homolog,

(c) an inactivation or deletion, or reduced expression or activity, ofaceE or aceE enzyme complex, and optionally

(d) a recombinant or modified idi gene to tune activity for higherterpene or terpenoid production.

Genes can be overexpressed by complementation with recombinant genes, orthe endogenous genes can be modified to alter expression, as disclosedelsewhere herein.

The bacterial strain is a bacteria selected from Escherichia spp.,Bacillus spp., Corynebacterium spp., Rhodobacter spp., Zymomonas spp.,Vibrio spp., and Pseudomonas spp. For example, the bacterial strain is aspecies selected from Escherichia coli, Bacillus subtilis,Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobactersphaeroides, Zymomonas mobilis, Vibrio natriegens, or Pseudomonasputida. In some embodiments, the bacterial strain is E. coli.

In various embodiments, upon culturing, HMBPP does not accumulate atmore than about 10 mg/g DCW, or in some embodiments does not accumulateat more than about 8 mg/g of DCW, or in some embodiments does notaccumulate at more than about 5 mg/g of DCW, or in some embodiments doesnot accumulate at more than about 4 mg/g DCW, or in some embodimentsdoes not accumulate at more than about 2 mg/g DCW. In some embodiments,HMBPP does not accumulate at more than about 1 mg/g DCW, or does notaccumulate at more than about 0.5 mg/g DCW, or more than about 0.2 mg/gDCW, or more than about 0.1 mg/g DCW.

In some embodiments, the bacterial strain expresses dxs, ispD, ispF, andidi as recombinant genes (e.g., as a complementation to wild-type MEPpathway enzymes), and which are optionally expressed as an operon. Insome embodiments, the bacterial strain expresses dxs, dxr, ispD, ispE,ispF, and idi as recombinant genes, which are optionally expressed as 1,2, or 3 individual operons. The recombinant genes of the MEP pathway areexpressed from one or more plasmids or are integrated into thechromosome, and the expressions are balanced to improve MEP carbon flux.Specifically, the bacterial cell may produce MEcPP as the predominantMEP metabolite in the extracellular medium.

The recombinant IspG and IspH genes may comprise one or more beneficialmutations, or may be an IspG or ispH ortholog having improved propertiesor activity, as described herein. Further, in various embodiments, theexpression of recombinant IspH is higher than the expression of therecombinant IspG, which can optionally be accomplished, at leastin-part, by positioning ispH before ispG in an operon. Thus, thebacterial strain may express ispH and ispG from the same operon (withispH positioned first), and under control of a strong promoter. Therecombinant IspG and IspH genes are expressed from a plasmid or areintegrated into the chromosome.

In some embodiments, the bacterial strain expresses a recombinant idigene, which is tuned to increase product, optionally by modifying thepromoter strength, gene copy number, position in an operon, or ribosomebinding site.

In some embodiments, the bacterial strain expresses a recombinant YdbKgene, which is integrated into the chromosome or expressed from aplasmid. The bacterial strain may further comprise an overexpression ofone or more of a flavodoxin, flavodoxin reductase, ferredoxin, andferredoxin reductase, such as Clostridium pasteurianum ferredoxin(Cp.fdx). In some embodiments, the strain expresses one or morenon-native fdx and/or fldA homologs. By way of example, the fdx homologmay be selected from Hm.fdx1 (Heliobacterium modesticaldum), Pa.fdx(Pseudomonas aeruginosa), Cv.fdx (Allochromatium vinosum), Ca.fdx(Clostridium acetobuylicum), Cp.fdx (Clostridium pasteurianum), Ev2.fdx(Ectothiorhodospira shaposhnikovii), Pplfdx (Pseudomonas putida) andPp2.fdx (Pseudomonas putida). In some embodiments, the fidA homologincludes one or more selected from Ec.fldA (E. coli), Ac.fldA2(Azotobacter chroococcum), Av.fldA2 (Azotobacter vinelandii), andBs.fldA (B. subtilis).

In some embodiments, the fdx homologs comprise a sequence that is atleast 60% identical to any one of SEQ ID NOs. 10 and 15-24. For example,the non-native fdx homologs can comprise a sequence that is at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, at least about 97%, at least about 98%, at leastabout 99%, or 100% identical to any of one of SEQ ID NOs. 10 and 15-24.

In some embodiments, the fldA homologs comprise a sequence that is atleast 60% identical to any one of SEQ ID NOs. 25-28. For example, thenon-native fidA homologs can comprise a sequence that is at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 97%, at least about 98%, at least about99%, or 100% identical to any of one of SEQ ID NOs. 25-28.

In some embodiments, the bacterial strain has overexpression orcomplementation with one or more PFOR and/or fpr and, optionally, one ormore of a flavodoxin (fldA), flavodoxin reductase, ferredoxin (fdx), andferredoxin reductase. By way of example, in some embodiments thebacterial strain includes Ec.ydhV (E. Coli) (SEQ ID NO: 33) and Ec.ydhY(E. Coli) (SEQ ID NO: 34); Ec.ydbK (E. Coli) (SEQ ID NO: 9) and Cp.fdx(Clostridium pasteurianum) (SEQ ID NO: 10); Ec.fpr (E. Coli) (SEQ ID NO:38) and Ec.fdx (E. Coli) (SEQ ID NO: 21); or Ec.fpr (E. Coli) (SEQ IDNO: 38) and Ec.fldA (E. coli) (SEQ ID NO: 27).

In some embodiments, the bacterial strain has overexpression orcomplementation with one or more PFOR, or a homolog thereof. By way ofexample, in some embodiments, the PFOR is selected from YdbK (SEQ ID NO:9), Scy.pfor (Synechocystis sp.) (SEQ ID NO: 29), Ki.pfor (Kluyveraintermedia) (SEQ ID NO: 30), Da.pfor (Desulfovibrio africanus) (SEQ IDNO: 31), Ns.pfor (Nostoc sp.) (SEQ ID NO: 32), Ec.ydhV (E. Coli) (SEQ IDNO: 33), Ga.pfor (Gilliamella apicola) (SEQ ID NO: 35), and Sco.pfor(Synechococcus sp.). In some embodiments, the PFOR is YdbK.

In some embodiments, the PFOR comprise a sequence that is at least 60%identical to any one of SEQ ID NOs. 29-35. For example, the PFOR cancomprise a sequence that is at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about97%, at least about 98%, at least about 99%, or 100% identical to any ofone of SEQ ID NOs. 29-35.

In some embodiments, the overexpression or complementation with PFORsuch as, e.g., YdbK, can result in improved performance throughexpression of electron carriers having a redox potential of about 400 to550 mV, or in some embodiments, in the range of about 400 to 500 mV, orin the range of about 400 to 475 mV. In some embodiment, the electroncarrier is ferrodoxin, flavodoxin, or NADPH. By way of example, in someembodiments, the electron carrier is Cv.fdx (Allochromatium vinosum).

In some embodiments, the bacterial strain has overexpression orcomplementation with one or more fpr homologs. By way of example, insome embodiments, the fpr homolog is selected from Ns.fpr (Nostoc sp.)(SEQ ID NO: 36), Sco.fpr (Synechococcus sp.) (SEQ ID NO: 37), and Ec.fpr(E. Coli) (SEQ ID NO: 38).

In some embodiments, the fpr comprise a sequence that is at least 60%identical to any one of SEQ ID NOs. 36-38. For example, the fpr cancomprise a sequence that is at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about97%, at least about 98%, at least about 99%, or 100% identical to any ofone of SEQ ID NOs: 36-38.

In some embodiments, the E. coli contains a deletion of one or moregenes selected from: pgrR, mppA, ynaI, insH-4, ynaJ, uspE, fnr, ogt,abgT, abgB, abgA, abgR, mcaS, isrA, smrA, ydaM, ydaN, fnrS, C0343, dbpA,REP115, ttcA, intR, ydaQ, ydaC, ralA, raR, recT, recE, racC, ydaE, andkilR.

The expression of the recombinant pgpB and/or nudB can be tuned toprovide higher product titer, optionally by varying the promoterstrength, gene copy number, position in an operon, and/or ribosomebinding site. In some embodiments, the recombinant pgpB and/or nudB isexpressed under control of a weak or intermediate strength promoter. Therecombinant pgpB or nudB is integrated into the chromosome or expressedfrom a plasmid.

In various embodiments, the bacterial strain produces a terpene orterpenoid product that comprises at least one of Amorphadiene,Artemisinic acid, Artemisinin, Bisabolol, Bisabolene, alpha-Sinensal,beta-Thujone, Camphor, Carveol, Carvone, Cineole, Citral, Citronellal,Cubebol, Farnesene, Geraniol, Limonene, Menthol, Menthone, Myrcene,Nootkatone, Nootkatol, Patchouli, Piperitone, Rose oxide, Sabinene,Steviol, Steviol glycoside (including Rebaudioside D or Rebaudioside M),Taxadiene, Thymol, and Valencene.

Aspects and embodiments of the invention are further demonstrated belowwith reference to the following Examples.

EXAMPLES Example 1: IspG/IspH Expression Tuning

Conclusions

Overexpression and balancing of MEP pathway genes can result in morecarbon entering the MEP pathway, and can shift that carbon ‘downstream’from DOXP and MEP to MEcPP. Modifying the expression of ispG and/or ispHmight further convert MEcPP to HMBPP to IPP.

In fact, increasing expression of both ispG and ispH significantlyincreased titers of terpene and terpenoid products. However, increasingexpression of just ispG or ispH alone did not improve titer.Overexpression of ispG alone resulted in growth defects, andoverexpression of ispH alone didn't significantly improve titer, but didconvert HMBPP to IPP. The effects of ispG overexpression could berelated to the observation that HMBPP is not found extracellularly, butis found 100% intracellularly. Since the molecule does not appear to betransported out of the cell, it may act as a feedback molecule,providing a hard stop on the MEP pathway. For example, if the pool ofHMBPP gets above a certain size, the pathway shuts down. Alternatively,or additional, HMBPP may be toxic at certain levels, which is consistentwith the observation of the impact on IspG overexpression on cellgrowth.

Thus, the balance of activity between ispG and ispH is important toprevent HMBPP imbalance and accumulation. In some situations, less ismore; that is, strongest overexpression of MEP genes can start to hurtproductivity.

In summary, overexpressing ispG and ispH together, where one or both ofispG or ispH are wild-type or mutated/engineered, in a properly balancedconfiguration, prevents HMBPP accumulation from becoming toxic andpushes carbon through the MEP pathway to IPP, DMAPP, and the downstreamterpene and terpenoid products.

Description of Experimental Results

FIG. 2 shows that increasing expression of IspH alone or IspH and IspGtogether improve terpenoid product titers in production strains that arealready engineered to increase the amount of carbon entering the MEPpathway, but ispG alone decreases productivity. In this example, thecontrol strain is an E. coli strain with balanced overexpression of MEPpathway genes (but no additional copies of ispG or ispH), increasing theamount of carbon entering the MEP pathway. As shown, overexpression ofIspG decreases product titer about 15%, while overexpression of IspH atthe same expression strength increases product titer 17%. Overexpressionof both IspG and IspH more than triples product titer.

The data also shows that a ispG/ispH ratio that favors more H enzymeresults in even more improved flux through the MEP pathway relative to astrain favoring the ispG side of the ratio. IspG and ispH are expressedhere in operon format, and thus the second gene in the operon will havea lower expression level than the first. Thus, ispH/ispG operon showedsignificantly more product titer than ispG/ispH.

IspG and ispH work sequentially to convert MEcPP to HMBPP, then to IPP.Increasing ispG will accumulate a larger HMBPP pool, while increasingispH will shrink the HMBPP pool as it is converted to IPP. The fact thatIspG alone decreases productivity, while ispH alone increases it,strongly suggests that accumulation of HMBPP has a negative feedbackeffect on the MEP pathway. When both IspG and IspH are overexpressed, weenhance the rate of both HMBPP formation and consumption, whichsignificantly improves flux through the MEP pathway to the targetterpenoid. However, even in this enhanced flux regime, the balance ofIspG to IspH is critical, since a slight favoring of ispH over ispG canfurther improve productivity by 25%, to nearly 4× the titers of a parentstrain having wild-type expression of IspG and IspH.

Increasing IspG and/or IspH expression in the modified productionstrains with enhanced MEP pathways impacts on the MEP productdistribution pattern (FIG. 3). The majority of products are DOX, ME, andMEcPP (FIG. 3, upper panel), followed by DOXP and MEP (FIG. 3, middlepanel), and HMBPP (FIG. 3, lower panel). In these panels, totalextracellular and intracellular metabolites are extracted from cultures(broth plus cells), such that the reported concentration is relative tovolume of extract.

Increasing IspG or IspH alone increases the conversion rate of MEcPP anddecreases the pool size (upper panel), even though IspH increasesproduct titer and IspG loses product titer. A significant differencebetween the two variants is however apparent with the HMBPPconcentration (lower panel), where IspG alone increases it 2.5× over thecontrol, while IspH alone decreases it 20%. This accumulation of HMBPPcould be feeding back on the MEP pathway and shutting down theenhancement of flux. HMBPP accumulates to very low levels (nMconcentration), and 100% of it is found intracellularly.

Increasing ispG and ispH expression together, in either operon order,can be seen to enable complete conversion of the remaining DOX, anddecreases the ME pool size. Moreover, a IspG/IspH ratio that favors moreIspH is capable of improved conversion of MEcPP (and improved producttiter) compared to a strain favoring IspG.

The proportion of each individual MEP metabolite found inside or outsidethe cell (‘Intra’ vs ‘Extra’) is shown in FIG. 4. These values do notreflect absolute abundance, as shown in FIG. 3, there is far more DOX intotal that there is HMBPP. While DOX is 100% extracellular, HMBPP is100% intracellular. The strain profiled here is the ‘Control+ispH/ispG’top performing strain from FIGS. 2 and 3. DOXP/DOX, MEP/ME, and MEcPPaccumulate almost entirely in the extracellular medium, while CDP-ME,CDP-MEP, HMBPP, IPP/DMAPP, and FPP are observed 100% intracelluarly. Thepercentage of each metabolite found intracellularly is shown at the topof the graph.

Uncompensated ispG upregulation causes a significant drop in cellgrowth, as determined by UV absorbance at 600 nm (FIG. 5). While somechanges to final cell density are observed in a strain compensated withispH or ispH and ispG together, the variation is not significant.

To determine HMBPP accumulation, HMBPP can be expressed in terms of drycell weight (DCW). For example, using a strain with balanced ispGHexpression:

[HMBPP]=0.42 ug/mL in 0.35 mL sampled culture

[OD600]=12.69

Assumption: 1 OD600=0.4 g−DCW/L=0.4 mg−DCW/mL

HMBPP yield=[(0.42 ug/mL)*(0.35 mL)]/[(12.69*0.4 mg−DCW/mL)*0.35 mL)]

In this example, HMBPP=0.0827 ug/mg DCW or 0.0827 mg/g DCW.

Example 2: pgpB and nudB Overexpression

Conclusions

Installing an alternate ‘product’ pull by overexpressing genes such aspgpB and nudB can pull even more flux through the MEP pathway (though tonon-target products), or could even replace the various downstreamterpenoid pathways to create a tool to engineer a ‘universal chassis’(i.e., a strain that can have any terpenoid downstream transformed intoit and be quickly optimized for commercial production).

Carbon can be pulled through the MEP pathway to create alternateproducts that will pool outside the cell. PgpB dephosphorylates FPP tofarnesol (FOH), and nudB dephosphorylates IPP and DMAPP to isoprenol(3-methyl-3-buten-1-ol) and prenol (3-methyl-2-buten-1-ol),respectively. Enhancing transport of these products outside the cellprevents buildup of IPP, DMAPP, and FPP; which like HMBPP, can feedbackand exert control on the MEP pathway. IPP inhibits growth and feedbackinhibits Dxs. See Cordoba, Salmi & Leon (2009) J. Exp. Bot. 60, 10,2933-2943. FPP feedback inhibits IspF-MEP complex, which itself isformed when MEP binds and enhances IspF activity in a feed-forwardmanner. Bitok & Meyers (2012) ACS Chem. Biol. 2012, 7, 1702-1710. Theseproducts accumulate outside the cell and, like the intermediates in theMEP pathway, can be used to track C-flux through the MEP pathway viaLC/MS metabolomics quantitation.

By constitutively expressing an additional copy of pgpB, carbon fluxthrough the MEP pathway can be improved, and a slow growth phenotypeameliorated in a strain that has MEP genes overexpressed but noadditional downstream pathway to pull all that carbon through toproduct. In effect, the downstream ‘pull’ becomes the conversion of FPPto farnesol, which is exported outside the cell. Similarly, constitutiveexpression of mdB should result in IPP and DMAPP pools beingincreasingly redirected to isoprenol and prenol extracellular products.

Further modulating the expression levels of MEP pathway genes in thepresence of overexpressed pgpB or nudB can significantly impact the MEPflux and carbon distribution through the pathway. The increase ordecrease in farnesol, prenol, or isoprenol product can be inverselycorrelated with MEcPP level.

Description of Experimental Results Overexpression of PgpB can triplefarnesol titers in strains engineered to enhance flux through the MEPpathway, but without a downstream terpenoid product pathway installed(FIG. 6). The control strain has additional copies of dxs, dxr, ispD,ispF, ispE, ispG, ispH, and idi under varying levels of constitutiveexpression, and also has YdbK overexpressed (Example 4). The controlaccumulates moderate amounts of farnesol, presumably as ‘spill-over’from too much FPP accumulation, which feeds back on the pathway, andsuffers from markedly slower growth compared to wild-type. When PgpB isoverexpressed in this strain, the excess FPP is more efficientlyconverted to farnesol (preventing feedback control) and the flux iseffectively pulled through the MEP pathway.

However, too much PgpB expression (the ‘+++’ condition) seems tonegatively impact the total flux through to farnesol, with lower titerand smaller fold-change observed, on average. Some potential reasons forthis result include: (1) too hard a pull from the PgpB is straining theMEP pathway's ability to keep up with FPP demand, especially fromrequired competing products; or (2) since PgpB is known todephosphorylate multiple targets in vivo, including an essentialmembrane phospholipid, a high expression level for PgpB could be havingunintended negative consequences on cell health.

Increasing and tuning expression of IspG′ and/or IspH in a strain thatproduces farnesol can improve product titer (FIG. 7). In this example,the IspG′ enzyme is an engineered version with higher activity thanwild-type. The control strain has additional copies of dxs, dxr, ispD,ispF, ispE, ispG, ispH, and idi, as well as additional copies of YdbKand pgpB. Additional copies of ispH and/or ispG′ are integrated into thestrains under increasing promoter strength (+, ++, +++).

When IspH is overexpressed (FIG. 7, panel A), no significant change inproduct titer is observed. While increasing the amount of IspH is goingto improve conversion of HMBPP to IPP, there is no additional IspG′ toprovide that additional HMBPP. The MEcPP pool mediated by IspG activitybecomes the rate-limiting step in the pathway. However, when IspG′ isoverexpressed in addition to IspH (FIG. 7, Panel B), we see asignificant increase in the farnesol product titer. In this situation,the additional HMBPP enabled by the additional copy of IspG′ is rapidlyconverted by the increased IspH level to IPP, preventing HMBPP fromaccumulating and feeding back on the pathway.

It is clear that the balance between IspG and IspH is critical. The datashows IspG/H being expressed in operon format, such that the second genein the operon will have a lower expression level than the first. Whenthe gene order in the operon for ispG′ and ispH is switched (i.e.,ispH+ispG′ versus ispG′+ispH) and thus changing the expression ratio ofIspG′/IspH, we see opposite trends in the data. When the ratio favorsIspH over IspG′ (B), an increasing promoter strength results in steadilyincreasing product titer. However, when the ratio favors IspG′ over IspH(C), the excess HMBPP that can be created by this imbalanced pathwaysteadily accumulates as promoter strength increases, resulting in lessand less product improvement and slower growth.

Increase in farnesol product titer can be accompanied by a decrease inMEcPP pool size, though it depends on the ratio of IspG and IspH (FIG.8). As seen in FIG. 7, additional copies of IspG′ and IspH in farnesolproducing strains can improve farnesol product titer up to 2.5-fold.When only IspH is upregulated without additional IspG (FIG. 8, Panel A),titer did not change significantly, nor did MEcPP. MEcPP does decreasemoderately, likely due to MEcPP being pulled downstream as HMBPP is moreefficiently converted to IPP by the extra IspH enzyme. When relativelymore IspH was expressed than IspG′ (FIG. 8, Panel B), as promoterstrength increases, farnesol product titer increased. MEcPP converselydecreases as it is consumed by a balanced pathway that distributes theflux to the desired end product.

However, even though a non-optimal ratio favoring IspG′ over IspH canimprove MEcPP conversion through HMBPP to IPP and improve farnesolproduct titer, eventually the imbalance is too severe for the E. colistrain to tolerate and the product improvement disappears, while evenmore MEcPP accumulates and is trapped in the MEP pathway intermediatecarbon pool.

Example 3: Idi Expression Tuning

Conclusions

Idi enzyme catalyzes the reversible isomerization of IPP to DMAPP. Sinceevery desired terpenoid product or undesired MEP side-product (e.g.,UPP) uses one DMAPP and varying numbers of IPP, the ratio between thetwo precursors could have a fundamental impact on strain productivity.For example, 1 FPP=1 DMAPP+2 IPP, whereas 1 UPP=1 FPP+8 IPP (or 1DMAPP+10 IPP). Therefore, an optimal ratio for FPPS to produce FPP is2:1 IPP:DMAPP, but 10:1 for UPP. Thus, varying the ratio of IPP:DMAPP byvarying idi expression will have an impact on the production of thedesired terpenoid relative to other undesired products from the MEPpathway.

Description of Experimental Results Idi was complemented in differentstrains producing product A or B. Cells were cultured in 96-round-wellculture plates at 37° C. for 48 hrs at 280 RPM in custom media withglucose as carbon source. Idi was expressed from a pBAC under anIPTG-inducible promoter. Strain 1 already has dxs, dxr, ispD, ispF,ispE, idi, FPPS, and YdbK overexpressed, while Strains 2 and 3 furtherhave ispH and a mutant version of IspG overexpressed in addition.Conversely, Strain 4 has the same enzymes overexpressed but under a verydifferent expression regime.

While Idi overexpression increases product titer in a strain that doesnot overexpress ispGH, it decreases titer in two strains that do,indicating that the balance between IPP and DMAPP controlled by Idi canbe tuned up or down depending on the needs of the downstream pathway(FIG. 9). However, Strain 4 more than doubles titer with idicomplementation. The same genes are overexpressed in this strain, butthe balance between the expression of the MEP genes is very different.

Example 4: YdbK Overexpression

Conclusions

YdbK is predicted to function as a pyruvate:flavodoxin oxidoreductaseand/or pyruvate synthase. The oxidoreductase is thought to oxidizepyruvate to acetyl-CoA, reducing ferredoxin, which can then supplyelectrons to the MEP pathway, especially to support the stronglyupregulated IspG and IspH enzymes that contain Fe—S clusters. YdbKoverexpression has been shown for hydrogen (H₂) production (Akhtar M K &Jones P R (2014), Cofactor engineering for enhancing the flux ofmetabolic pathways.” Frontiers in Bioeng. and Biotech.), but not forterpenoid production.

The product titer of terpene Product A doubled in these strains. TheFe—S clusters are better supported by the extra YdbK cofactor, and theiractivity improves. Product titer goes up, and when the MEP metabolitesare profiled, we see an increased conversion of MEcPP, similar to whatis observed when the control strain further adds another copy ofispH-ispG′ operon.

On the other hand, when a Product B strain that didn't have IspG/Hoverexpressed relative to WT, was complemented with YdbK, the Product Btiter went down. When IspG/H was increased in this strain, YdbKcomplementation did improve Product B titer, suggesting that YdbKexpression has to be carefully balanced with IspG/H expression (which,in turn must be carefully balanced for H/G ratio).

Additionally, extra electron-carrying or transferring cofactors wereadded on top of the YdbK overexpression to see if we can further improvetiters. In some experiments, YdbK plus fdx (ferredoxin) from Clostridiumpasteurianum improved productivity somewhat.

Description of Experimental Results

An additional copy of E. coli YdbK gene is integrated into chromosome orexpressed on a plasmid (specifically a single-copy pBAC, or multi-copyplasmids), under control of constitutive or inducible promoters.Additionally, copies of native or non-native recombinant electronacceptor/donors can also be overexpressed with YdbK, to capitalize onand utilize most efficiently the additional electrons made available forbiosynthesis.

Expressing an additional copy of YdbK under increasing promoter strengthcan improve terpenoid production. In this example, the control strainproduces terpenoid product A, and has additional copies of genes dxs,dxr, ispD, ispE, ispF, ispG′, ispH, and idi of the MEP pathway underdefined constitutive expression.

In this strain, adding an extra copy of ispH and ispG′ in operon format(such that the H/G′ ratio favors H) further increases the Product Atiter, indicating that these steps are limiting (FIG. 10, Panel A).Increasing these Fe—S cluster-containing genes clearly increases theconversion of MEcPP and lowers the concentration observed in culture(FIG. 10, Panel C).

When YdbK is complemented in the control strain, we see a gradedresponse to upregulation, where increasing expression sees increasingterpenoid production, up to a point—moving to stronger expressionresults in 50% less Product A in the +++YdbK strain (FIG. 10, Panel B).We see the same conversion of MEcPP occurring for these strains (FIG.10, Panel D), suggesting that YdbK is supporting enhanced IspG and/orIspH activity. Of note, the MEP metabolite profile of ++vs+++strainsdoesn't change significantly, but has ˜3× less product titer, suggestingthat some kind of feedback mechanisms has been activated. Given theobservations of work with IspG and IspH, it is possible that thisfeedback is due to HMBPP accumulation.

The improvement in terpenoid product titer from increasing YdbKexpression requires sufficient IspG and/or IspH to be manifested (FIG.11). In this example, Control A has additional copies of dxs, ispD,ispF, and idi of the MEP pathway, as well as rhyB deletion and iscoperon changes. Control B is Control A plus an additional integratedcopy of ispG′ and ispH in operon configuration (G′ first, such that theH/G ratio favors G), while Control C is Control A plus an additionalintegrated copy of ispH and ispG′ in operon configuration (H first, suchthat the H/G ratio favors H).

In Panel A, we see that complementing YdbK in the absence of IspG/Hupregulation decreases terpenoid Product B titer by about 25%. However,when you complement YdbK in strains with additional copies of ispG′-ispHor ispH-ispG′, we observed 18% and 27% improvement in terpenoid titers.Clearly, IspG and IspH must be overexpressed relative to WT MEP pathwayto see the benefit of YdbK.

Moreover, this data again highlights how important the expressionbalance between IspG and IspH can be for MEP pathway flux and terpenoidproductivity. In control B vs. C, the same enzymes are upregulated underthe same promoter strength—the difference lies in the order of genes inthe operon. The genes closest to the promoter will be expressed morestrongly than subsequent genes in the operon, such that the H/G enzymesratio favors IspG in Control B or IspH in Control C. Given this, weobserve that a ratio favoring H improves titer more so than one favoringG. Moreover, the improvement made possible by YdbK is enhanced in astrain favoring H. Thus, the balance between IspH and IspG is veryimportant to strain productivity.

Expressing fdx in addition to YdbK can further improve terpenoid titers(FIG. 12). In this example, the control strain produces terpenoidproduct A, and has additional copies of genes dxs, dxr, ispD, ispE,ispF, ispG′, ispH, and idi of the MEP pathway under defined constitutiveexpression.

As shown in FIG. 10, expressing an additional copy of YdbK underconstitutive expression increases the production of Product A.Complementing was attempted with three additional electronacceptors/donors, fldA, fldA and erpA (each from E. coli), or fdx fromClostridium pasteurianum (which may support 4Fe-4S clusters, as arefound in IspG and IspH, rather that 2Fe-2S clusters).

Adding another copy of fldA (flavodoxin) or fldA and erpA (essentialrespiratory protein A) in addition to YdbK did not further improveProduct A titer, but adding Clostridium pasteurianum fdx did improvetiters of Product A. Interestingly, while addition of YdbK results incomplete conversion of DOX/DOXP downstream to ME/MEP, further adding fdAcauses some carbon to pool upstream in the MEP pathway as DOX/DOXP.Adding erpA to the mix restores the profile. However, the MEP metaboliteprofile for the further enhanced ydbK+fdx strain is most similar toydbK+fldA, suggesting that optimum MEP flux will result from coordinatedbalancing of the MEP pathway gene expression as well as expression ofcritical electron donor/acceptors.

Example 5: Reducing PDH Conversion of Pyruvate to Acetyl-COA EnhancesYdbK Conversion of Pyruvate to Acetyl-COA

Increasing the reliance on YdbK for the conversion of pyruvate toacetyl-COA can improve the production of terpenes and/or terpenoidproducts by the engineered microbial strain because YdbK has a lowerredox potential (larger absolute number in Table 4) than the FMNhydroquinone/semiquinone couple in fidA. As such, YdbK is the preferredsource of electrons (not fpr/NADPH) by IspG and IspH.

Iron sulfur clusters (e.g., Fe₄S₄) in enzymes (such as IspG and IspH)utilize a wide range of reduction potentials, e.g., −200 to −800 mV.Blachly, et al., Inorganic Chemistry, 54(13): 6439-6461 (2015).

Reduction potentials for charging electron carriers YdbK and fpr aredisclosed in Tables 2 and 3, respectively, and reduction potentials fordischarging electron carries (e.g., YdbK and fpr) to IspG and IspH aredisclosed in Table 4. See McIver, et al., FEBS J, 257(3):577-85 (1998)and Lupton, et al., J. Bacteriol, 159: 843-9 (1984).

TABLE 2 Charging Electron Carrier YdbK ΔG⁰ (kcal/mol ε⁰ (mV) pyruvate)YdbK oxidation half reaction: pyruvate + CoA → acetyl-CoA + CO₂ + 2H⁺ +2e⁻ 540 (D. africanus) Potential electron carrier reduction halfreactions: 2 oxidized fldA + 2e⁻ + 2H⁺ → 2 semiquinone fldA −254 −13.2 2fdx 2Fe(III)²⁺ + 2e⁻ → 2 fdx Fe(III)²⁺Fe(II)¹⁺ −380 −7.4 2 semiquinonefldA + 2e⁻ + 2H⁺ → 2 hydroquinone −433 −4.9 fldA

TABLE 3 Charging Electron Carrier fpr ΔG⁰ (kcal/mol ε⁰ (mV) NADPH) Step1 NADPH oxidation half reaction: NADPH → NADP⁺ + 2e⁻ + H⁺ 320 (370 forNADPH/ NADP+ = 60) Potential fpr reduction half reactions: 2 oxidizedfpr + 2e⁻ + 2H⁺ → 2 −308 −0.6 semiquinone fpr 2 semiquinone fpr + 2e⁻ +2H⁺ → 2 −268 −2.4 hydroquinone fpr Step 2 Potential fpr oxidation halfreaction: semiquinone fpr → oxidized fpr + e⁻ + H⁺ 308 hydroquinone fpr→ semiquinone fpr + e⁻ + 268 H⁺ Potential electron carrier reductionhalf reactions: oxidized fldA + e⁻ + H⁺ → semiquinone −254 −2.5 or −0.6fldA

TABLE 4 Discharging Electron Carriers to IspG and IspH ε⁰ Potentialelectron carrier oxidation half reactions: (mV) Source 2 semiquinonefldA → 2 oxidized fldA + 2e⁻ + 2H⁺ 254 Fpr, YdbK 2 fdx Fe(III)²⁺Fe(II)¹⁺→ 2 fdx 2Fe(III)²⁺ + 2e⁻ 380 YdbK 2 hydroquinone fldA → 2 semiquinonefldA + 2e⁻ + 433 YdbK 2H⁺

The optimal activity of IspG was tested in vitro by using a range ofredox dyes. Xiao, et al., Biochemistry, 48(44):10483-10485 (2009). Theoptimal activity of IspG was tested with externally fed methyl viologen(=446 mV). The activity of IspG using fed methyl viologen (ε⁰=446 mV)was 20× greater than an in vitro fpr-fldA system.

IspH activity was 50× greater with methyl viologen (ε⁰=446 mV) and 100×greater with the externally fed dithionite-MDQ (ε⁰=490 mV). Xiao, etal., Journal of the American Chemical Society, 131(29): 9931-9933(2009).

It is hypothesized that the fldA semi qui none/hydroquinone couple thatis accessible by YdbK, but not fpr, is the preferable in vivo reductionsystem for IspG and IspH.

In order to increase a microbial strain's reliance on PFOR (e.g., YdbK)mediated conversion of pyruvate to acetyl-COA, PDH mediated conversionof pyruvate to acetyl-COA was reduced. See FIG. 15.

There are three known reactions in E. coli to convert pyruvate (PYR) toacetyl-CoA (AcCoA): pflB, PDH, and PFOR or YdbK.

Out of the three enzymes, PDH predominates and is a multi-enzyme complex(aceE-aceF-lpd), which consists of 24 subunits of pyruvate dehydrogenase(aceE), 24 subunits of lipoate acetyltransferase (aceF), and 12 subunitsof dihydrolipoate dehydrogenase (lpd). The net reaction of the PDHsystem, in addition to reducing NAD+, is the conversion of pyruvate intoAcCoA and CO₂, a key reaction of central metabolism because it linksglycolysis I, which generates pyruvate, to the TCA cycle, into which theAcCoA flows. During aerobic growth, PDH is an essential source of AcCoAto feed the TCA cycle and thereby to satisfy the cellular requirementsfor the precursor metabolites it forms. Mutant strains defective in thePDH complex require an exogenous source of acetate to meet thisrequirement.

pflB is only active in anaerobic condition. As such, it is not a primaryreaction to convert PYR to AcCoA under microaerobic and aerobicconditions.

In microbial strains with at least YdbK overexpression, PDH (see, e.g.,Example 4) is no longer essential since YdbK can be used to supplyAcCoA. To ensure the PYR to AcCoA step is mainly catalyzed by YdbK,which in turn supplies electrons to IspG and IspH, PDH activity wasreduced or eliminated through gene knockouts or knockdowns (e.g., bymutation).

Elimination of PDH Via Knockout of aceE

Four different E. coli strains engineered to produce four differentterpenoid products (indicated as Product B, Product C, Product D, andProduct E) were further engineered to knockout aceE (ΔaceE), whicheliminated PDH activity. Control strains were the same, but without theaceE knockout.

The data shows an increase in titer of each of the four terpenoidproducts through the deletion of aceE as compared to control. See FIGS.16A-D. Differences in fold-change improvement can largely be attributedto the biochemical characteristics (e.g., Km and k_(cat)) of thedifferent terpenoid synthase enzymes employed for the downstreampathway. Specifically, enzymes with lower synthase activity compared tothe Product D synthase (the most catalytically efficient) had slightlylower fold-change improvements, presumably due to the accumulation ofthe FPP substrate, which can accumulate and lead to either cell toxicityor feedback regulation on upstream components of the MEP pathway.

The data also shows a reduction in MEcPP concentrations in theextracellular broth (FIG. 16E) as compared to control, which confirmsthat carbon flux has been pushed through the IspG/H steps into product.See FIG. 1.

Knockdown of PDH Via Mutated aceE

Three E. coli strains, each of which were engineered to produce threedifferent terpenoid products (shown as Product B, Product C, and ProductD) were further engineered to express a mutated aceE (G267C; aceE mut),which resulted in reducing PDH activity. Control strains were the same,but did not have a mutated aceE.

Similar to the aceE knockout results, the data shows an increase intiter of each of the three terpenoid products in the microbial strainsexpressing mutated aceE as compared to control. See FIGS. 17A-C.

The data also shows a reduction in MEcPP concentrations in theextracellular broth (FIG. 17D) as compared to control, which confirmsthat carbon flux has been pushed through the IspG/H steps into product.See FIG. 1.

Example 6: Non-Native Electron Acceptors/Donors Increase YdbK-DependentIsoprenoid Production

When YdbK was overexpressed in E. coli, native ferredoxin (fdx) orflavodoxin (fldA) shuttled electrons to IspG and IspH (PYR/YdbK/fldA orfdx). E. coli engineered to produce Product B and overexpress YdbK wasfurther engineered to overexpress one of the following fdx homologs inTable 5 or fldA homologs in Table 6. The first seven fdx homologs are2[4Fe-4S] ferredoxins meaning they contain two 4Fe-4S iron-sulfurclusters that can have either the same or different redox potentials.For ferredoxin where the clusters differ in redox potential, given theredox potential of YdbK, we anticipate that in most cases cluster 1 willbe the relevant cluster. The remaining fdx homologs are a 2Fe-2Sferredoxin and a high potential 4Fe-4S ferredoxin, both of which containa single cluster. Control E. coli did not express any fdx or fdAhomologs.

TABLE 5 Fdx Homologs Cluster 1 Cluster 2 fdx (mV) (mV) Organism Hm.fdx1−480 −524 Heliobacterium modesticaldum Pa.fdx −475 −655 Pseudomonasaeruginosa Cv.fdx −467 −640 Allochromatium vinosum Cv.fdx_C57A −451 −590Synthetic Ec.yfhL −418 −675 E. coli Ca.fdx −400 −400 Clostridiumacetobutylicum Cp.fdx −390 −390 Clostridium pasteurianum Ev2.fdx +50 —Ectothiorhodospira shaposhnikovii Pp1.fdx — — Pseudomonas putida Pp2.fdx— — Pseudomonas putida

TABLE 6 FldA Homologs Semiquinone −> Hydroquinone −> oxidizedsemiquinone fldA (mV) (mV) Organism Ac.fldA2 −522 −133 Azotobacterchroococcum Av.fldA2 −483 −187 Azotobacter vinelandii Ec.fldA −433 −254E. coli Bs.fldA −382 −105 B. subtilis

The data shows that overexpression of certain fdx or fldA homologs in E.coli and overexpress YdbK had increased titers of terpenoid product(Product Bin this example) as compared to the empty vector control (emp)(e.g., H.fdx, Cv.fdx, Cv.fdxC57A, and Pa.fdx). FIG. 19A.

E. coli engineered to produce Product D and overexpress YdbK werefurther engineered to overexpress Cv.fdx. Similar to the previousresults, the data shows that overexpression of Cv.fdx in E. coliengineered to produce a terpenoid product (Product D in this example)and overexpress YdbK had increased titers of terpenoid product ascompared to control. FIG. 19B. Additionally, the data shows a reductionin MEcPP concentrations in the extracellular broth (FIG. 19C) ascompared to control, which confirms that carbon flux has been pushedthrough the IspG/H steps into product. See FIG. 1.

Example 7: Overexpression or Complementation with PFOR Homologs, FprHomologs, and/or fdx or fldA Homologs

E. coli engineered to produce Product F were further engineered tooverexpress at least one PFOR homologs or fpr homologs and, optionally,a fdx or fldA homolog as shown in Table 7.

TABLE 7 Group Homolog SEQ ID NO(s) 1 Scy.pfor (Synechocystis sp.) SEQ IDNO: 29 2 Ki.pfor (Khuyvera intermedia) SEQ ID NO: 30 3 Da.pfor(Desulfovibrio africanus) SEQ ID NO: 31 4 Sco.pfor (Synechococcus sp.) —5 Ec.ydhV and Ec.ydhY (E. Coli) SEQ ID NO: 33 and SEQ ID NO: 34 6Ga.pfor (Gilliamella apicola) SEQ ID NO: 35 7 Ec.ydbK (E. Coli) SEQ IDNO: 9 8 Ec.ydbK and Cp.fdx (E. Coli and SEQ ID NO: 9 and 10 C.pasteurianum) 9 Ns.fpr (Nostoc sp.) SEQ ID NO: 36 10 Sco.fpr(Synechococcus sp.) SEQ ID NO: 37 11 Ec.fpr and Ec.fdx (E. Coli) SEQ IDNO: 38 and SEQ ID NO: 21 12 Ec.fpr and Ec.fldA (E. Coli) SEQ ID NO: 38and SEQ ID NO: 27

The data shows that some bacterial strains engineered to express PFORand fpr homologs had increased titers of terpenoid product (Product F inthis example) as compared to empty vector control (CTRL) (e.g., Da.pfor(Desulfovibrio africanus) (SEQ ID NO: 31); Sco.pfor (Synechococcus sp.);Ga.pfor (Gilliamella apicola) (SEQ ID NO: 35); Ec.ydbk (E. Coli) (SEQ IDNO: 9); and Sco.fpr (Synechococcus sp.) (SEQ ID NO: 37). See FIG. 20.

The data also shows that bacterial strains engineered to overexpress atleast one PFOR homolog and a fdx had increased titers of terpenoidproduct (Product F) as compare to empty vector control (CTRL) (e.g.,Ec.ydhV/Ec.ydhY; E. coli (SEQ ID NO: 33 and SEQ ID NO: 34, respectively)and Ec.ydbK/Cp.fdx; E. coli (SEQ ID NO: 9 and 10, respectively)). SeeFIG. 20.

Additionally, the data shows that bacterial strains engineered tooverexpress at least one fpr homologs and either fdx or fldA hadincreased titers of terpenoid product (Product F) as compare to emptyvector control (CTRL) (e.g., Ec.fpr/Ec.fdx; E. coli (SEQ ID NO: 38 andSEQ ID NO: 21, respectively) and Ec.fpr/Ec.fldA; E. coli (SEQ ID NO: 38and SEQ ID NO: 27, respectively)). See FIG. 20.

SEQUENCES (E. coli IspG) SEQ ID NO: 1MHNQAPIQRRKSTRIYVGNVPIDGAPIAVQSMTNTRTTDVEATVNQIKALERVGADIVRVSVPTMDAAEAFKLIKQQVNVPLVADIHFDYRIALKVAEYGVDCLRINPGNIGNEERIRMVVDCARDKNIPIRIGVNAGSLEKDLQEKYGEPTPQALLESAMRHVDHLDRLNFDQFKVSVKASDVFLAVESYRLLAKQIDQPLHLGITEAGGARSGAVKSAIGLGLLLSEGIGDTLRVSLAADPVEEIKVGFDILKSLRIRSRGINFIACPTCSRQEFDVIGTVNALEQRLEDIITPMDVSIIGCVVNGPGEALVSTLGVTGGNKKSGLYEDGVRKDRLDNNDMIDQLEARIRAKASQLDEARRIDVQQVEK (E. coli IspH) SEQ ID NO: 2MQILLANPRGFCAGVDRAISIVENALAIYGAPIYVRHEVVHNRYVVDSLRERGAIFIEQISEVPDGAILIFSAHGVSQAVRNEAKSRDLTVFDATCPLVTKVHMEVARASRRGEESILIGHAGHPEVEGTMGQYSNPEGGMYLVESPDDVWKLTVKNEEKLSFMTQTTLSVDDTSDVIDALRKRFPKIVGPRKDDICYATTNRQEAVRALAEQAEVVLVVGSKNSSNSNRLAELAQRMGKRAFLIDDAKDIQEEWVKEVKCVGVTAGASAPDILVQNVVARLQQLGGGEAIPLEGREENIVFEVPKELRVDIREVD (E. coli Dxs) SEQ ID NO: 3MSFDIAKYPTLALVDSTQELRLLPKESLPKLCDELRRYLLDSVSRSSGHFASGLGTVELTVALHYVYNTPFDQLIWDVGHQAYPHKILTGRRDKIGTIRQKGGLHPFPWRGESEYDVLSVGHSSTSISAGIGIAVAAEKEGKNRRTVCVIGDGAITAGMAFEAMNHAGDIRPDMLVILNDNEMSISENVGALNNHLAQLLSGKLYSSLREGGKKVFSGVPPIKELLKRTEEHIKGMVVPGTLFEELGFNYIGPVDGHDVLGLITTLKNMRDLKGPQFLHIMTKKGRGYEPAEKDPITFHAVPKFDPSSGCLPKSSGGLPSYSKIFGDWLCETAAKDNKLMAITPAMREGSGMVEFSRKFPDRYFDVAIAEQHAVTFAAGLAIGGYKPIVAIYSTIFLQRAYDQVLHDVAIQKLPVLFAIDRAGIVGADGQTHQGAFDLSYLRCIPEMVIMTPSDENECRQMLYTGYHYNDGPSAVRYPRGNAVGVELTPLEKLPIGKGIVKRRGEKLAILNFGTLMPEAAKVAESLNATLVDMRFVKPLDEALILEMEMAASHEALVTVEENAIMGGAGSGVNEVLMAHRKPVPVLNIGLPDFFIPQGTQEEMRAELGLDAAG MEAKIKAWLA(E. coli Dxr) SEQ ID NO: 4MKQLTILGSTGSIGCSTLDVVRHNPEHFRVVALVAGKNVTRMVEQCLEFSPRYAVMDDEASAKLLKTMLQQQGSRTEVLSGQQAACDMAALEDVDQVMAAIVGAAGLLPTLAAIRAGKTILLANKESLVTCGRLFMDAVKQSKAQLLPVDSEHNAIFQSLPQPIQHNLGYADLEQNGVVSILLTGSGGPFRETPLRDLATMTPDQACRHPNWSMGRKISVDSATMMNKGLEYIEARWLFNASASQMEVLIHPQSVIHSMVRYQDGSVLAQLGEPDMRTPIAHTMAWPNRVNSGVKPLDFCKLSALTFAAPDYDRYPCLKLAMEAFEQGQAATTALNAANEITVAAFLAQQIRTDIAALNLSVLEKMDMREPQCVDDVLSVD ANAREVARKEVMRLAS(E. coli IspD) SEQ ID NO: 5MATTHLDVCAVVPAAGFGRRMQTECPKQYLSIGNQTILEHSVHALLAHPRVKRVVIAISPGDSRFAQLPLANHPQITVVDGGDERADSVLAGLKAAGDAQWVLHDAARPCLHQDDLARLLALSETSRTGGLAAPVRDTMKRAEPGKNAINHTVDRNGLWHALTPQFFPRELLHDCLTRALNEGATITDEASALEYCGFHPQLVEGRADNIKVTRPEDLALAEFYLTRTIHQENT (E. coli IspE) SEQ ID NO: 6MRTQWPSPAKLNLFLYITGQRADGYHTLQTLFQFLDYGDTISIELRDDGDIRLLTPVEGVEHEDNLINTRAARLEMKTAADSGRLPTGSGANISIDKRLPMGGGLGGGSSNAATVLVALNHLWQCGLSMDELAEMGLTLGADVPVFVRGHAAFAEGVGEILTPVDPPEKWYLVAHPGVSIPTPVIFKDPELPRNTPKRSIETLLKCEFSNDCEVIARKRFREVDAVLSWLLEYAPSRLTGTGACVFAEFDTESEARQVLEQAPEWLNGFVAKGANLSPLHRAML(E. coli IspF) SEQ ID NO: 7MRIGHGFDVHAFGGECIPIIIGGVRIPYEKGLLAHSDGDVALHALTDALLGAAALGDIGKLFPDTDPAFKGADSRELLREAWRRIQAKGYTLGNVDVTIIAQAPKMLPHIPQMRVFIAEDLGCHMDDVNVKATTTEKLGFTGRGEGIACEAVALLIKATK (E. coli Idi)SEQ ID NO: 8 MQTEHVILLNAQGVPTGTLEKYAAHTADTRLHLAFSSWLFNAKGQLLVTRRALSKKAWPGVWTNSVCGHPQLGESNEDAVIRRCRYELGVEITPPESIYPDFRYRATDPSGIVENEVCPVFAARTTSALQINDDEVMDYQWCDLAADVLHGIDATPWAFSPWMVMQATNREARKRLSAFTQLK (E. coli YdbK) SEQ ID NO: 9MITIDGNGAVASVAFRTSEVIAIYPITSSTMAEQADAWAGNGLKNVWGDTPRVVEMQSEAGAIATVHGALQTGALSTSFTSSQGLLLMIPTLYKLAGELTPFVLHVAARTVATHALSIFGDHSDVMAVRQTGCAMLCAANVQEAQDFALISQIATLKSRVPFIHFFDGFRTSHEINKIVPLADDTILDLMPQVEIDAHRARALNPEHPVIRGTSANPDTYFQSREATNPWYNAVYDHVEQAMNDFSAATGRQYQPFEYYGHPQAERVIILMGSAIGTCEEVVDELLTRGEKVGVLKVRLYRPFSAKHLLQALPGSVRSVAVLDRTKEPGAQAEPLYLDVMTALAEAFNNGERETLPRVIGGRYGLSSKEFGPDCVLAVFAELNAAKPKARFTVGIYDDVTNLSLPLPENTLPNSAKLEALFYGLGSDGSVSATKNNIKIIGNSTPWYAQGYFVYDSKKAGGLTVSHLRVSEQPIRSAYLISQADFVGCHQLQFIDKYQMAERLKPGGIFLLNTPYSADEVWSRLPQEVQAVLNQKKARFYVINAAKIARECGLAARINTVMQMAFFHLTQILPGDSALAELQGAIAKSYSSKGQDLVERNWQALALARESVEEVPLQPVNPHSANRPPVVSDAAPDFVNKTVTAAMLAGLGDALPVSALPPDGTWPMGTTRWEKRNIAEEIPIWKEELCTQCNHCVAACPHSAIRAKVVPPEAMENAPASLHSLDVKSRDMRGQKYVLQVAPEDCTGCNLCVEVCPAKDRQNPEIKAINMMSRLEHVEEEKINYDFGLNLPEIDRSKLERIDIRTSQLITPLFEYSGACSGCGETPYIKLLTQLYGDRMLIANATGCSSIYGGNLPSTPYTTDANGRGPAWANSLFEDNAEFGLGFRLTVDQHRVRVLRLLDQFADKIPAELLTALKSDATPEVRREQVAALRQQLNDVAEAHELLRDADALVEKSIWLIGGDGWAYDIGFGGLDHVLSLTENVNILVLDTQCYSNTGGQASKATPLGAVTKFGEHGKRKARKDLGVSMMMYGHVYVAQISLGAQLNQTVKAIQEAEANTGPSLIIAYSPCEEHGYDLALSHDQMRQLTATGFWPLYRFDPRRADEGKLPLALDSRPPSEAPEETLLHEQRFRRLNSQQPEVAEQLWKDAAADLQKRYDFL AQMAGKAEKSNTD(Clostridium pasteurianum fdx; Cp.fdx) SEQ ID NO: 10MAYKIADSCVSCGACASECPVNAISQGDSIFVIDADTCIDCGNCANVCPVGAPVQE (E. coli PgpB)SEQ ID NO: 11 MRSIARRTAVGAALLLVMPVAVWISGWRWQPGEQSWLLKAAFWVTETVTQPWGVITHLILFGWFLWCLRFRIKAAFVLFAILAAAILVGQGVKSWIKDKVQEPRPFVIWLEKTHHIPVDEFYTLKRAERGNLVKEQLAEEKNIPQYLRSHWQKETGFAFPSGHTMFAASWALLAVGLLWPRRRTLTIAILLVWATGVMGSRLLLGMHWPRDLVVATLISWALVAVATWLAQRICGPLTPPAEENREIAQREQES (E. coli NudB) SEQ ID NO: 12MKDKVYKRPVSILVVIYAQDTKRVLMLQRRDDPDFWQSVTGSVEEGETAPQAAMREVKEEVTIDVVAEQLTLIDCQRTVEFEIFSHLRHRYAPGVTRNTESWFCLALPHERQIVFTEHLAYKWLDAPAAAALTKSWSNRQAIEQFVINAA(E. coli Shine Dalgarno sequence) SEQ ID NO: 13 AGGAGG(Methanococcus maripaludis GAPOR) SEQ ID NO: 14MNILIDGSRQNYEELEESEFPISFGINLHTKQETWKYDAFDEKNLFCFGKGILPIIGGHRLIFSFRSPLWDGFHFSAMGGAGYTFKDTGIQNVAITGKCEVPTVIVLNGEEDKLKIEFMPFTEEITDIYEFNDKIIDLFKEKNYRAFLVGPASKTTNMGGIYSQTIRNGKIVEGSEDWAARGGGGSVLYQAHNVLGVVFFGKKTPEKNLKEIVEEHYNKPYTKVVLEHTEKYRYSEEKKTGGTFGNNYHVTMELTPVFNWRMPFIDKNKRMKLHKKIIEYFVNRFDEEAIETKNWTNCGEPCPVVCKKYRKGLHVDYEPYEANGPCIGVFDIYAADKVVHTIDKLGFDAIEFGNLCSWTFELLDNGMLKPEEVGIEKPVFDISNFENDEDILKNSMHNAEQAVKLAEIIAFQTNEFGKICKSGTRRAGKILNEKYPDRIKDKKFEDFGVYDSFGERGQISPTMYWAIGNFMPYLIQGKYLTHYQCGVFLEPEELAELSVKNSIEEITLENLGICRFHRKWVTPIIEKLVKEMSDVNLNEESMELFKKIAKYDSNIGCPEMESERVKELIIAGAFEFENEKWSKEFENGNFDEYIKRVLEKYSELLEIDWKLKE(Heliobacterium modesticaldum , Hm.fdx1) SEQ ID NO: 15MAYKITDACTACGACMDGCCVGAIVEGKKYSITSDCVDCGVCADKCPVDAIIPG(Pseudomonas aeruginosa, Pa.fdx) SEQ ID NO: 16MSLKITDDCINCDVCEPECPNGAISQGEEIYVIDPNLCTECVGHYDEPQCQQVCPVDCIPLDDANVESKDQLMEKYRKITGKA (Allochromatium vinosum, Cv.fdx)SEQ ID NO: 17 MALMITDECINCDVCEPECPNGAISQGDETYVIEPSLCTECVGHYETSQCVEVCPVDCIIKDPSHEETEDELRAKYERITGEG (Cv.fdx_C57A) SEQ ID NO: 18MALMITDECINCDVCEPECPNGAISQGDETYVIEPSLCTECVGHYETSQCVEVCPVDAIIKDPSHEETEDELRAKYERITGEG (E. coli, Ec.yfhL) SEQ ID NO; 19MALLITKKCINCDMCEPECPNEAISMGDHIYEINSDKCTECVGHYETPTCQKVCPIPNTIVKDPAHVETEEQLWDKFVLMHHADKI (Clostridium acetobutylicum, Ca.fdx)SEQ ID NO: 20 MAYKITDACVSCGSCASECPVSAISQGDTQFVIDADTCIECGNCANVCPVGAPVQE(E. coli, Ec.fdx) SEQ ID NO: 21MPKIVILPHQDLCPDGAVLEANSGETILDAALRNGIEIEHACEKSCACTTCHCIVREGFDSLPESSEQEDDMLDKAWGLEPESRLSCQARVTDEDLVVEIPRYTINHAREH(Ectothiorhodospira shaposhnikovii, Ev2.fdx) SEQ ID NO: 22MERLSEDDPAAQALEYRHDASSVQHPAYEEGQTCLNCLLYTDASAQDWGPCSVF PGKLVSANGWCTAWVAR(Pseudomonas pulida, Pp1.fdx) SEQ ID NO: 23MSLIITDDCINCDVCEPECPNAAISQGEEIYVIDPNLCTQCVGHYDEPQCQQVCPVDCIPLDEAHPETHDELMEKYKRITGKA (Pseudomonas putida, Pp1.fdx) SEQ ID NO: 24MSLIITDDCINCDVCEPECPNEAISQGEEIYVIDPNLCTQCVGHYPQCQQVCPVDCIPLDEAHPETEEELMAKYRRIT (Azotobacter vinelandii fldA2; Av.fldA2)SEQ ID NO: 25 MAKIGLFFGSNTGKTRKVAKSIKKRFDDETMSDALNVNRVSAEDFAQYQFLILGTPTLGEGELPGLSSDCENESWEEFLPKIEGLDFSGKTVALFGLGDQVGYPENYLDALGELYSFFKDRGAKIVGSWSTDGYEFESSEAVVDGKFVGLALDLDNQSGKTDERVA AWLAQIAPEFGLSL(Azotobacter chroococcum fldA2; Ac.fldA2) SEQ ID NO: 26MAKIGLFFGSNTGKTRKVAKSIKKRFDDETMSDAVNVNRVSAEDFAQYQFLILGTPTLGEGELPGLSSDCENESWEEFLPKIEGLDFSGKTVALFGLGDQVGYPENFLDAMGELHSFFTERGAKVVGAWSTDGYEFEGSTAVVDGKFVGLALDLDNQSGKTDERV AAWLAQIAPEFGLSL(E. coli, Ec.fldA) SEQ ID NO: 27MAITGIFFGSDTGNTENLAXMIQKQLGKDVADVHDIAKSSKEDLEAYDILLLGIPTWYYGEAQCDWDDFFPTLEEIDFNGKLVALFGCGDQEDYAEYGCDALGTIRDIIEPRGATIVGHWPTAGYHFEASKGLADDDHFVGLAIDEDRQPELTAERVEKWVKQISEE LHLDEILNA(B. subtilis, Bs.fldA) SEQ ID NO: 28MAKALITYASMSGNTEDIAFIIKDTLQEYELDIDCVEINDMDASCLTSYDYVLIGTYTWGDGDLPYEAEDFFEENTKQIQLNGLKTACFGSGDYSYPKFCEAVNLFNVMLQEAGAAVYQETLKIELAPETDEDVESCRAFARGFLAWADYMNKEKIHVS(Synechocystis sp., Scy.pfor) SEQ ID NO: 29MSLPTYATLDGNEAVARVAYLLSEVIAIYPITPSSPMGEWSDAWAAEHRPNLWGTVPLVVEMQSEGGAAGTVHGALQSGALTTTFTASQGLMLMLPNMHKIAGELTAMVLHVAARSLAAQGLSIFGDHSDVMAARNTGFAMLSSNSVQEAHDFALIATATSFATRIPGLHFFDGFRTSHEEQKIELLPQEVLRGLIKDEDVLAHRGRALTPDRPKLRGTAQNPDVYFQARETVNPFYASYPNVLEQVMEQFGQLTGRHYRPYEYCGHPEAERVIVLMGSGAETAQETVDFLTAQGEKVGLLKVRLYRPFAGDRLVNALPKTVQKIAVLDRCKEPGSIGEPLYQDVLTAFFEAGMMPKIIGGRYGLSSKEFTPAMVKGVLDHLNQTNPKNHFTVGINDDLSHTSIDYDPSFSTEADSVVRAIFYGLGSDGTVGANKNSIKIIGEDTDNYAQGYFVYDSKKSGSVTVSHLRFGPNPILSTYLISQANFVACHQWEFLEQFEVLEPAVDGGVFLNSPYGPEEIWREFPRKVQQEIIDKNLKVYTINANDVARDAGMGRRTNTVMQTCFFALAGVLPREEAIAKIKQSVQKTYGKKGQEIVEMNIKAVDSTLAHLYEVSVPETVSDDAPAMRPVVPDNAPVFVREVLGKIMARQGDDLPVSALPCDGTYPTATTQWEKRNVGREIPVWDPDVCVQCGKCVIVCPHAVIRGKVYEEAELANAPVSFKFTNAKDHDWQGSKFTIQVAPEDCTGCGICVDVCPAKNKSQPRLRAINMAPQLPLREQERENWDFFLDLPNPDRLSLNLNKISHQQMQEPLFEFSGACAGCGETPYLKLVSQLFGDRMLVANATGCSSIYGGNLPTTPWAQNAEGRGPAWSNSLFEDNAEFGLGFRVAIDKQTEFAGELLKTFAGELGDSLVSEILNNAQTTEADIFEQRQLVEQVKQRLQNLETPQAQMFLSVADYLVKKSVWIIGGDGWAYDIGYGGLDHVLASGRNVNILVMDTEVYSNTGGQASKATPRAAVAKFAAGGKPSPKKDLGLMAMTYGNVYVASIANIGAKNEQSIKAFMEAEAYPGVSLIIAYSHCIAHGINMTTAMNHQKELVDSGRWLLYRYNPLLADEGKNPLQLDMGSPKVAIDKTVYSENRFAMLTRSQPEEAKRLMKLAQGDVNTRWAMYEYLAKRSLGGEINGNNHGVSPSPEVIAKSV(Kluyvera intermedia, Ki.pfor) SEQ ID NO: 30MSGKMKTMDGNAAAAWISYAFTDVAAIYPITPSTPMAENVDEWTAQGKKNLFGQPVRLMEMQSEAGAAGAVHGALQAGALTTTYTASQGLLLMIPNLYKIAGELLPGVFHVSARALATNSLNIFGDHQDVMAVRQTGCAMLAENNVQQVMDLSAVAHLSAIKGRVPFINFFDGFRTSHEIQKIEVLEHEALAPLLDQEALNLFRRNALNPDHPVIRGTAQNPDIYFQEREASNRFYQALPDIVEGYMAEIRITGREYHLFDYYGSPDAEQIIIAMGSVCDTIQEVVDAMIDSGEKVGLVSVHLFRPFSLAHFMAKIPASVKRIAVLDRTKEPGAQAEPLCLDVKNAFYHHDNPPLIVGGRYALGGKDVLPGHIVSVFENLKKPLPMDGFTVGIFDDVTHTSLPVPAYDIHVSREGITACKFWGLGSDGTVSANKNAIKIIGDNTSMFAQAYFAYDSKKSGGITMSHLRFGKRPITSPYLIHNADFIACSQQSYVDKYDLLDGINPGGIFLLNCTWFGEEVERHLPNKMKRIIARQGVRFYTLNAVDIARKLGLGGRFNAILMQAAFFKLTDIIDAKTASEHLKKAVAKSYGSKGQNVVDMNNAAIDLGMDALQEIIVPDHWAYVEEEANNDGKLMPDFIRNILEPMNRQNGDKLPVSAFLGMEDGTFPPGTAAWEKRGIAMQVPVWQPEGCTQCNQCAFICPHAAIRPALLSSEEREAAPVALLSKVAQGAKHYEYHLAVSPLDCSGCGNCVDICPSKGKALAMKPLDSQRHMVPVWDHALALAPKENPFSKATVKGCQFEPPLLEFSGACAGCGETPYARLITQLFGDRMMIANATGCSSIWGASAPSIPWTTNHKGQGPAWANSLFEDNAEFGLGMMLGGRAIREQLASDAASVLERPLHPDLQQALRDWLEHKDLGEGTRARAEKLSALLAAEKGDDDLLNRLYQNQDYFTKRSQWIFGGDGWAYDIGFGGLDHVLASGEDVNILVFDTEVYSNTGGQSSKSTPVAAIAKFAAEGKRTRKKDLGMMAVSYGNVYVAQVAMGADKAQTLRAIAEAEAWPGPSLVIAYAACINHGLKAGMGRSISEAKRAVEAGYWHLWRYNPQLLAKGKNPFILDSEEPEESFRDFLMGEVRYASLGRTSPEVADSLFAQTEQDAKDRYAQYRRLAGE (Desulfovibrio africanus, Da.pfor) SEQ ID NO: 31MGKKMMTTDGNTATAHVAYAMSEVAAIYPITPSSTMGEEADDWAAQGRKNIFGQTLTIREMQSEAGAAGAVHGALAAGALTTTFTASQGLLLMIPNMYKISGELLPGVFHVTARAIAAHALSIFGDHQDIYAARQTGFAMLASSSVQEAHDMALVAHLAAIESNVPFMHFFDGFRTSHEIQKIEVLDYADMASLVNQKALAEFRAKSMNPEHPHVRGTAQNPDINTQGREAANPYYLKVPGIVAEYMQKVAALTGRSYKLFDYVGAPDAERVIVSMGSSCETIEEVINHLAAKGDKIGLIKVRLYRPFVSEAFFAALPASAKVITVLDRTKEPGAPGDPLYLDVCSAFVERGEAMPKILAGRYGLGSKEFSPAMVKSVYDNMSGAKKNHFTVGIEDDVFGTSLPVDNAFADTTPKGTIQCQFWGLGADGTVGANKQAIKIIGDNTDLFAQGYFSYDSKKSGGITISHLRFGEKPIQSTYLVNRADYVACHNPAYVGIYDILEGIKDGGTFVLNSPWSSLEDMDKHLPSGIKRTIANKKLKFYNIDAVKIATDVGLGGRINMIMQTAFFKLAGVLPFEKAVDLLKKSIHKAYGKKGEKIVKMNTDAVDQAVTSLQEFKYPASWKDAPAETKAEPKTNEFFKNVVKPILTQQGDKLPVSAFEADGRFPLGTSQFEKRGVAINVPQWVPENCIQCNQCAFVCPHSAILPVLAKEEELVGAPANFTALEAKGKELKGYKFRIQINTLDCMGCGNCADICPPKEKALVMQPLDTQRDAQVPNLEYAARIPVKSEVLPRDSLKGSQFQEPLMEFSGACSGCGETPYVRVITQLFGERMFIANATGCSSIWGASAPSMPYKTNSLGQGPAWGNSLFEDAAEYGFGMNMSMFARRTHLADLAAKALESDASGDVKEALQGWLAGKNDPIKSKEYGDKLKKLLAGQKDGLLGQIAAMSDLYTKKSVWIFGGDGWAYDIGYGGLDHVLASGEDVNVFVMDTEVYSNTGGQSSKATPTGAVAKFAAAGKRTGKKDLARMVMTYGYVYVATVSMGYSKQQFLKVLKEAESFPGPSLVIAYATCINQGLRKGMGKSQDVMNTAVKSGYWPLFRYDPRLAAQGKNPFQLDSKAPDGSVEEFLMAQNRFAVLDRSFPEDAKRLRAQVAHELDVRFKELERMAATNIFESFAPAGGKADGSVDFGEGAEFCTRDDTPMMARPDSGEACDQNRAGTSEQQGDLSKRTKK (Nostoc sp., Ns.pfor) SEQ ID NO. 32MSQTFATIDGNEAVARVAYKLNEVIAIYPITPSSAMGEWADAWMAEGRPNLWGTVPSVVQMQSEGGAAGAVHGALQTGSLSTTFTASQGLLLMIPNLYKIGGELTSMVVHVAARSLATHALSIFGDHSDVMAARGTGFAMLCSASVQESHDFALIAHAATLDTRVSFLHFFDGFRTSHEVQKVELLADDDVRSLINEDKIFAHRARALTPDSPLLRGTAQNPDVFFQAREGANPYYNACPAIVQGIMDKFGERTGRYYQIYEYHGASDADRLIIIMGSGCETVHETVDYLNARGEKVGVLKVRLFRPWDVERFVQALPHSVQAIAVLDRTKEPGSAGEPLYQDVVTAIHEGWVNKNNSPVPSPQSPVPKIIGGRYGLSSKEFTPAMVKAVFDNLAQATPKNHFTIGINDDVTHTSLEYDPSFSTEPDNVVRAMFYGLGSDGTVGANKNSIKIIGEGTDNYAQGYFVYDSKKSGSMTVSHLRFGSQPIRSTYLIDQANFIGCHHWGFLERIEVLNAAAHGATILLNSPYNAATVWENLPLKVRLQILDKQLKLYVINANQVARDSGMGGRINTIMQVCFFALAGVLPEVQAIAKIKQAIEKTYGKKGVEVVRMNLQAVDQTLENLHEVKIPIEEKGKWIDEEALLSNQSPFSTSAPKFVRDVLGKIMVWQGDDLPVSTLPPDGTFPTGTAKWEKRNVAQEIPVWDTDICVQCSKCVMVCPHAAIRAKVYQPSELENAPPTFKSVDAKDRDFANQKFTIQVAPEDCTGCAICVNVCPAKNKSEPSLKAINMANQLPLREQERDNWDFFLNLPNPDRRNLKLNQIRQQQLQEPLFEFSGACAGCGETPYVKLLTQLFGDRSVIANATGCSSIYGGNLPTTPWTKNNDGRGPAWSNSLFEDNAEFGFGYRLSLDKQAEFAAELLQQFSTEVGDNLVDSILKAPQKTEADIWEQRQRIELLKQQLDKIPTFDPNLKSKIQNLKSLADYLVKKSVWIIGGDGWAYDIDFGGIDHVIASGRNVNILVMDTEVYSNTGGQSSKATPKAAVAKFAASGKPAQKKDMGLMAMNYGNVYVASVALGAKDDQTLKAFLEAEAFDGPSIIIAYSHCIAHGINMTTGMNQQKALVESGRWLLYRYNPLLQEQGKNPLQLDMRSPTQSVEQSMYQENRFKMLTKSKPEVAKQLLEQAQAEVDARWQMYQYLASR (E. coli, Ec.ydhV)SEQ ID NO: 33 MANGWTGNILRVNLTTGNITLEDSSKSFVGGMGFGYKIMYDEVPPGTKPFDEANKLVFATGPLTGSGAPCSSRVNITSLSTFTKGNLVVDAHMGGFFAAQMKFAGYDVIIIEGKAKSPVWLKIKDDKVSLEKADFLWGKGTRATTEEICRLTSPETCVAAIGQAGENLVPLSGMLNSRNHSGGAGTGAIMGSKNLKAIAVEGTKGVNIADRQEMKRLNDYMMTELIGANNNHVVPSTPQSWAEYSDPKSRWTARKGLFWGAAEGGPIETGEIPPGNQNTVGFRTYKSVFDLGPAAEKYTVKMSGCHSCPIRCMTQMNIPRVKEFGVPSTGGNTCVANFVHTTIFPNGPKDFEDKDDGRVIGNLVGLNLFDDYGLWCNYGQLHRDFTYCYSKGVFKRVLPAEEYAEIRWDQLEAGDVNFIKDFYYRLAHRVGELSHLADGSYAIAERWNLGEEYWGYAKNKLWSPFGYPVHHANEASAQVGSIVNCMFNRDCMTHTHINFIGSGLPLKLQREVAKELFGSEDAYDETKNYTPINDAKIKYAKWSLLRVCLHNAVTLCNWVWPMTVSPLKSRNYRGDLALEAKFFKAITGEEMTQEKLDLAAERIFTLHRAYTVKLMQTKDMRNEHDLICSWVFDKDPQIPVFTEGTDKMDRDDMHASLTMFYKEMGWDPQLGCPTRETLQRIGLEDIAADIAAHNLLPA (E. coli, Ec.ydhY)SEQ ID NO: 34MNPVDRPLLDIGLTRLEFLRISGKGLAGLTIAPAIILSILGCKQEDIDSGTVGLINTPKGVLVTQRARCTGCHRCEISCTNFNDGSVGTFFSRIKIHRNYFFGDNGVGSGGGLYGDLNYTADTCRQCKEPQCMNVCPIGAITWQQKEGCITVDHKRCIGCSACTTACPWMMATVNTESKKSSKCVLEGECANACPTGALKIIEWKDITV (Gilliamella apicola, Ga.pfor)SEQ ID NO: 35 MIISDANSAVSSVAYRANEVIAIYPITPSSSMAEQASTWAEFDKPNVFGDIPRVVEMQSEAGAIATVHGALMTGALATSFTSSQGLLLMIPSLYKIAGELTPFVLHVAARTVATHALSIFGDHSDVMSVRQTGFAMLCSSSVQEAQLALISQIASFKSRIPFVHFFDGFRTSHEVNKIYPLSDEDIHDLLPHEAIKAYRSRALTPDKPMIRGTSANPDTYFQCREAINSYYDNAYQHVVDAMTDFEKQTGRKYQPFEYYGASDAERIIVIMGSGASTSKEVIDYLLKENQKVGVVIVRLFRPFSAQHLLAVIPDSVKKIAVLDRTKEPGAQAEPLYLDIMTAFAESLSRGERNTIPQIVGGRYGLSSKEFDPRSVLGIFNELSLEKPRPRFTVGIYDDITGLSLPLPDKTIPQKSALEALFYGLGSDGTVSATKNNIKIIGDSSPFYVQGYFVYDSKKAGGLTTSHLRVNLDPIDSPYLITSAHFIGCHQDQFIDKYQIVDKLKNDGIFLLNTPYNKDEIWHRLPKEVQVQLIKKRAHFYIINAAKIARECNLGARINTVMQAAFFHLSDIFKNDFSISQLKEVIAKSYSSKGQELVENNWKALDLAITSLEQIPLNCVDQSSPSMPPIVPNNAPDFVKTVTATMLAGLGDSLPVSAFPPDGAWPTGTTKWEKRNIAEEIPIWKSELCTQCNHCAVACPHAAIRAKVVEPDAMLNAPDTLESLEVKARDMKGQRYVLQVAPEDCTGCNLCVEVCPSRDRNNFDIKAINMQPRIDNLDTQRVNFEFFSALPDRD1KSLDRIDIRTSQLITPLFEYSGACAGCGETPYIKLLTQLYGDHLAIANATGCSSIYGGNLPSTPYTTDRSGRGPAWANSLFEDNAEFALGYRITYNQHRKRALRLLDHLAGEISPEIVITLQSSDATIAEKRTQVDLLREQLKHIDSAEAKELLEDTNYLIDKSVWAIGGDGWAYDIGFGGLDHVMSLTDNVNILVLDTQCYSNTGGQQSKATPMGAVSKFADLGKHKARKDLGVSIMMYGHVYVAQVALGSQLNQTLKALQEAEAYDGPSLNIAYSPCEEHGYDLAKSHEQMKDLVKSGFWPLYRYDPRRSAEGKPGLVLDSKSPNSEALSSILLKEQRFRRLETLEPTVANILHERSTKMVESKYRFLQMLSSYSDIETPPDS(Nostoc sp., Ns.fpr) SEQ ID NO: 36MSNQGAFDGAANVESGSRVFVYEVVGMRQNEETDQTNYPIRKSGSVFIRVPYNRMNQEMQRITRLGGKIVTIQTVSALQQLNGRTTIATVTDASSEIAKSEGNGKATPVKTDSGAKAFAKPPAEEQLKKKDNKGNTMTQAKAKHADVPVNLYRPNAPFIGKVISNEPINKEGGIGIVQHIKFDLTGGNLKYIEGQSIGIIPPGVDKNGKPEKLRLYSIASTRHGDDVDDKTISLCVRQLEYKHPESGETVYGVCSTYLTHIEPGSEVKITGPVGKEMLLPDDPEANVIMLATGTGIAPMRTYLWRMFKDAERAANPEYQFKGFSWLVFGVPTTPNILYKEELEEIQQKYPDNFRLTYAISREQKNPQGGRMYIQDRVAEHADELWQLIKNQKTHTYICGLRGMEEGIDAALSAAAAKEGVTWSDYQKDLKKAGRWHVETY(Synechococcus sp., Sco.fpr) SEQ ID NO: 37MYGITSTANSTGNQSYANRLFIYEVVGLGGDGRNENSLVRKSGTTFITVPYARMNQEMQRITKLGGKIVSIRPAEDAAQIVSEGQSSAQASAQSPMASSTKIVHPKTTDTSVPVNIYRPKTPFLGKCIENYELVDEGGSGTVRHVTFDISEGDLRYLEGQSIGIIPPGEDKNGKPHKLRLYSIASTRHGDMEDNKTVSLCVRQLEYQDPESGETVYGVCSTYLCNLPVGTDDVKITGPVGKEMLLPDDEDATVVMLATGTGIAPFRAFLWRMFKEQHEDYKFKGKAWLIFGVPYTANILYKDDFEKMAAENPDNFRLTYAISREQKTADGGKVYVQSRVSEYADELFEMIQKPNTHVYMCGLKGMQPPIDETFTAEAEKRGLNWEEM RRSMKKEHRWHVEVY(E. coli, Ec.fpr) SEQ ID NO: 38MADWVTGKVTKVQNWTDALFSLTVHAPVLPFTAGQFTKLGLEIDGERVQRAYSYVNSPDNPDLEFYLVTVPDGKLSPRLAALKPGDEVQVVSEAAGFFVLDEVPHCETLWMLATGTAIGPYLSILQLGKDLDRFKNLVLVHAARYAADLSYLPLMQELEKRYEGKLRIQTVVSRETAAGSLTGRIPALIESGELESTIGLPMNKETSHVMLCGNPQMVRDTQQLLKETRQMTKHLRRRPGHMTAEHYW

1. A method for production of a terpene or terpenoid product,comprising: providing a bacterial strain that produces isopentenyldiphosphate (IPP) and dimethylallyl diphosphate (DMAPP) through anupstream methylerythritol phosphate pathway (MEP pathway) and convertsthe IPP and DMAPP to a terpene or terpenoid product through a downstreamsynthesis pathway; wherein IspG and IspH are overexpressed in thebacterial strain such that IspG activity and IspH activity are balancedto provide increased carbon flux to 1-hydroxy-2-methyl-2-(E)-butenyl4-diphosphate (HMBPP) intermediate, while preventing accumulation ofHMBPP at an amount that feeds back and reduces MEP pathway flux andterpene or terpenoid productivity; the strain optionally comprising oneor more genetic modifications that enhance supply and/or transfer ofelectrons through the MEP pathway and/or to terpene and terpenoidproducts, and culturing the bacterial strain to produce the terpene orterpenoid product.
 2. The method of claim 1, wherein the bacterialstrain is a bacteria selected from Escherichia spp., Bacillus spp.,Corynebacterium spp., Rhodobacter spp., Zymomonas spp., Vibrio spp., andPseudomonas spp.
 3. The method of claim 2, wherein the bacterial strainis a species selected from Escherichia coli, Bacillus subtilis,Corynebacterium glutamicum, Rhodobacter capsulatus, Rhodobactersphaeroides, Zymomonas mobilis, Vibrio natriegens, or Pseudomonasputida.
 4. The method of claim 3, wherein the bacterial strain is E.coli. 5.-7. (canceled)
 8. The method of claim 1, wherein IspG and IspHare overexpressed by introducing recombinant ispG and ispH genes intothe bacterial strain.
 9. The method of claim 8, wherein the recombinantIspG and/or IspH enzymes comprises one or more beneficial mutations, oris an ortholog having improved properties or activity under conditionsused for culturing.
 10. The method of claim 1, wherein the activityand/or expression of recombinant IspH is higher than the activity and/orexpression of the recombinant IspG.
 11. The method of claim 10, whereinthe expression of the recombinant IspH is higher than the expression ofthe recombinant IspG. 12.-13. (canceled)
 14. The method of claim 1,wherein expression of the recombinant IspG and IspH enzymes are balancedby modifying the promoter strength, gene copy number, and/or ribosomebinding site sequence of the ispG and/or ispH recombinant genes. 15.-16.(canceled)
 17. The method of claim 1, wherein the expression or activityof a recombinant idi gene is tuned to increase terpene or terpenoidproduction.
 18. The method of claim 17, wherein the expression of therecombinant idi gene is tuned by modifying the promoter strength, genecopy number, position in an operon, or ribosome binding site. 19.(canceled)
 20. The method of claim 1, wherein the bacterial straincomprises one or more genetic modifications that enhance supply and/ortransfer of electrons through the MEP pathway and/or to terpene andterpenoid products, and which optionally oxidizes pyruvate and/orreduces ferredoxin. 21.-34. (canceled)
 35. A method for draining excesscarbon flux from the MEP pathway in a bacterial cell, comprising:providing a bacterial strain that produces isopentenyl diphosphate (IPP)and dimethylallyl diphosphate (DMAPP) through an upstreammethylerythritol phosphate pathway (MEP pathway) and optionally convertsthe IPP and DMAPP to a terpene or terpenoid product through a downstreamsynthesis pathway; wherein the bacterial strain overexpresses one ormore MEP pathway genes and overexpresses PgpB and/or NudB, culturing thebacterial strain to produce MEP carbon. 36.-59. (canceled)
 60. Themethod of claim 1, wherein the bacterial strain has reduced oreliminated PDH mediated conversion of pyruvate to acetyl-CoA. 61.-99.(canceled)
 100. A method for making an industrial or consumer product,comprising, incorporating the terpene or terpenoid made according to themethod of claim 1 into said industrial or consumer product.
 101. Themethod of claim 100, wherein the industrial or consumer product is aflavor product, a fragrance product, a sweetener, a cosmetic, a cleaningproduct, a detergent or soap, or a pest control product.
 102. The methodof claim 100, wherein the industrial or consumer product is a food,beverage, texturant, pharmaceutical, tobacco product, nutraceutical,oral hygiene product, or cosmetic product.
 103. The method of claim 1,wherein the bacterial strain or cell overexpresses one or more ofgeranyl diphosphate synthase (GPPS), farnesyl diphosphate synthase(FPPS), and geranylgeranyl diphosphate synthase (GGPPS).
 104. Abacterial strain having one or more genetic modifications that increaseproduction of products from isopentenyl diphosphate (IPP) anddimethylallyl diphosphate (DMAPP) precursors; the bacterial strainproducing isopentenyl diphosphate (IPP) and dimethylallyl diphosphate(DMAPP) through an MEP pathway, and the MEP pathway comprising one ormore overexpressed genes; the strain converting the IPP and DMAPP to aterpene or terpenoid product through a downstream synthesis pathway;wherein said genetic modifications comprise: (a) recombinant or modifiedIspG and IspH enzymes, the IspG and IspH enzymes having balancedexpression or activity to prevent accumulation of HMBPP intermediate,(b) a recombinant or modified gene encoding an enzyme that enhancessupply and/or transfer of electrons through the MEP pathway and/or toterpene or terpenoid products, which is optionally an overexpression ofa YdbK gene and optionally with a non-native fdx and/or fldA homolog,(c) an inactivation or deletion, or reduced expression or activity, ofaceE or aceE enzyme complex, and optionally (d) a recombinant ormodified idi gene to tune activity for higher terpene or terpenoidproduction. 105.-136. (canceled)